Front. Transplant. Frontiers in Transplantation Front. Transplant. 2813-2440 Frontiers Media S.A. 10.3389/frtra.2025.1614849 Transplantation Review Long-term storage, cryopreservation, and culture of isolated human islets: a systematic review ChenAustin R. 1 2 ChanskyJoshua 1 BurkeJacqueline A. 1 * Department of Biomedical Engineering, Northwestern University, Chicago, IL, United States Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

Edited by: Tatsuya Kin, University of Alberta Hospital, Canada

Reviewed by: Anil Kharga, University of Pennsylvania, United States

Julie Kerr-Conte, Université de Lille, France

 Correspondence: Jacqueline A. Burke jacqueline.burke@northwestern.edu 08082025 2025 41614849 19042025 04072025 © 2025 Chen, Chansky and Burke. 2025Chen, Chansky and Burke

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 Introduction

Islet transplantation offers a potential curative treatment for patients with type 1 diabetes (T1D). To make this therapy widely available, a stable supply chain of human islets is essential. Developing techniques like cryopreservation and culture for long-term islet storage, or islet banking, with minimal functional loss would strengthen this supply chain. This study provides a systematic review of the current methods for long-term human islet storage.

Methods

A search strategy and query were developed according to the PICO framework. We included studies published on PubMed, Embase, and Web of Science from inception until August 2024.

Results

6,945 studies were screened with 47 meeting criteria for full text extraction. The primary outcomes recorded were measures of islet viability and glucose stimulated insulin secretion. Optimization of culture parameters such as temperature, medium selection, and scaffolds can extend islet viability and function.

Discussion

Recent studies on human islet cryopreservation report promising results for long-term storage; however, the field remains underexplored. Several cytoprotective supplements with potential utility across both culture and cryopreservation conditions have also been reviewed. Although long-term islet storage has been a critical focus since the advent of the Edmonton protocol, the literature lacks the rigor needed to drive clinical translation. Notably, we observe substantial variability in experimental design and reported outcomes, which complicates meaningful comparison between interventions.

 type 1 diabetes (T1D) islet transplantation human islets islet storage cryopreservation culture techniques islet viability glucose-stimulated insulin secretion (GSIS) T35 5T35DK126628-043-SRA-2023-1389-S-B2-SRA-2023-1452-S-BThe Northwestern Summer Research Program for Medical StudentsStrategic Research AgreementDiversifying Diabetes Research Talent in Academia Award section-at-acceptanceCell and Stem Cell Transplantation Introduction

In June 2023, the Food and Drug Administration approved Lantidra, the first allogeneic pancreatic islet therapy, for treating patients with type 1 diabetes (T1D) experiencing severe hypoglycemia (1). While patients receiving Lantidra must undergo immunosuppressive therapy, this approval signals a potential future where islet transplantation could become a curative option for all T1D patients. However, two major obstacles must be overcome to realize this future fully: the need for immunosuppression and the limited supply of islets. Here, we focus on the challenge of islet shortage. Current potential sources of islets include human, xenogeneic, and stem cell-derived islets. Each of these options presents unique challenges. Immunosuppressive protocols have yet to be optimized to enable clinical xenogeneic islet transplants. Stem cell-derived islets, while promising, also carry risks, including the potential for teratoma formation (2). At this point in time, human islets are the most suitable for transplant. However, the current supply of human islets cannot meet the demand of all existing and newly diagnosed patients.

Approximately 7,000 pancreases are donated each year in the United States (3). The timing and geographical constraints of deceased donor transplantations limit this number. With 64,000 newly diagnosed cases of T1D every year (4), this supply of pancreata is not enough for curative treatment of new T1D patients, much less the existing population of 2 million. In addition, it is unclear whether each pancreas would supply the recommended 5,000 islet equivalents (IEQ)/kg for insulin independence in a patient (5). Islet isolation after pancreas harvesting leads to a 15%–50% reduction in islet mass and function (6). Further loss of islet viability occurs during transplantation and engraftment. If islets could be stored for extended periods, the geographic pool of viable recipients could be expanded, and islets could be banked to build a sufficient supply of necessary IEQs for each patient. However, the clinical standard for islet preservation only makes them viable for transplantation for a few days after isolation. Possible solutions to long-term storage include optimized culture conditions and cryopreservation. Islet culture occurs in an enriched medium at physiologic temperatures (37°C) (7). Islets die quickly in culture due to inadequate oxygen delivery to the center of the cell clusters (8). Cryopreservation involves freezing islets to ultra-low temperatures (−196°C) using liquid nitrogen (9). Ultra-low temperatures drastically reduce the biological and chemical activity of cells, limiting energy consumption and cell death (10). Optimization of both methods is measured by islet death and the loss of islet function. In this systematic review, the current state of long-term human islet storage, via culture and cryopreservation is summarized. In addition, cytoprotective supplements, such as antioxidants and oxygen carriers, and in vivo experimentation with stored human islets are reviewed.

 Methods

The PRISMA 2020 guidelines and PICO framework were utilized to develop this systematic review (11). The PICO or population, intervention, control, outcome framework is a widely used approach to boolean query of scientific databases (12). Specifying key terms for each component of PICO ensures accurate knowledge representation of a research question that will capture all available studies that are related (13). A PICO framework search query was developed focused on the research question “What are the best techniques for ex vivo human islet cell preservation as measured by islet viability and glucose sensitive insulin secretion?” was developed in coordination with Northwestern University Galter Library Systematic Review Services. The population was identified as adult human islets, intervention was identified as islet preservation by cryopreservation or culture, a control was defined as freshly isolated human islets but was not used in the search, and outcome was identified as glucose-stimulated insulin release (GSIS) or islet viability. The search was limited to studies using human islets only to maximize the clinical relevance of this review as non-human islet models have significantly different architecture and biochemistry (14, 15). The full PICO-based query is reported in Table 1. This query was used to extract studies from PubMed, Embase, and Web of Science.

Distribution of preservation methods assessed by in vitro experiments by decade.

Stacked bar chart showing the number of in vitro studies by decade, categorized by preservation method. Culture studies increased from 1990–2019 and decreased in 2020–present. Cryopreservation studies were consistently fewer, peaking between 2000–2019.

Deduplication and screening of query results was carried out using the Rayyan platform (16). Query records were deduplicated by manual review of text with exact Title, Author, and Year matches by ARC. Inclusion and exclusion criteria specified in Table 2 were used by ARC and JAB to screen abstracts. All possible inclusions were reviewed again by ARC. Conflicts were resolved via discussion between ARC and JAB.

PICO framework and MeSH terms utilized to query PubMed, Embase, and Web of Science.

PICO Keywords and MeSH Terms used
Population Keywords: islet-cell* OR islet-culture* OR pancreatic-islet* OR islets-of-langerhans OR langerhans-islet* OR insulin-secreting-cell* OR beta-cell* OR alpha-cell* OR islet-spheroid* MeSH: "Islets of Langerhans"[Mesh] OR "Insulin-Secreting Cells"[Mesh]
Intervention Keywords: cryoprotect* OR preserv* OR cryopreservation OR cultur* OR slow-cooling OR vitrification OR suspension-culture* OR embedding OR encapsulation OR scaffolds OR bioreactor* OR microencapsulation OR islet-seeding OR islet-transplantation* OR islet-graft* OR islet-isolation OR islet-banking MeSH: "Islets of Langerhans Transplantation"[Mesh] OR "Preservation, Biological"[Mesh] OR "Tissue Preservation"[Mesh] OR "Cell Culture Techniques"[Mesh] OR "Organ Culture Techniques"[Mesh] OR "Culture Media"[Mesh]
Control None identified
Outcomes Keywords: glucose-stimulated-insulin-secretion* OR glucose-stimulated-insulin-release OR islet-equivalent* OR islet-purity OR islet-viability OR islet-death OR islet-volume OR GSIS OR number-of-islet* OR islet-number* OR count OR potency OR diabetic-nude-mouse-bioassay* OR membrane-integrity OR bioenergetic-status OR oxygen-consumption-rate* OR islet-morpholog* OR islet-yield OR islet-diameter OR cell-line-authentication OR cell-size OR cell-shape OR cell-survival MeSH: "Insulin Secretion"[Mesh] OR "Cell Line Authentication"[Mesh] OR "Cell Count"[Mesh] OR "Cell Size"[Mesh] OR "Cell Shape"[Mesh] OR "Cell Survival"[Mesh]

Abstract screening inclusion and exclusion criteria.

Include Exclude

English language

Full manuscript

Research article

Includes assessment of adult human islets following preservation via cryopreservation OR culture

Languages other than English

Poster/conference proceeding/presentation

Review paper

Does NOT include assessment of adult human islets

ONLY includes assessment of animal, fetal pancreata, AND/OR induced pluripotent stem cell derived islets

Does NOT involve preservation via cryopreservation or culture

Full text retrieval and extraction were performed by ARC and JC. Eligibility of the full text was evaluated based on the criteria in Table 3. Alongside measurements of viability and GSIS, methods and associated storage time and temperature were summarized for each study and associated treatment groups. Due to lack of standardized measures of islet viability and GSIS, units were collected for each study.

Full text extraction inclusion and exclusion criteria.

Include Exclude

Quantifies islet viability OR glucose stimulate insulin secretion (GSIS) following preservation

Describes method used to quantify islet viability OR GSIS

Does NOT quantify islet viability AND GSIS following preservation

Does NOT describe method used to quantify islet viability OR GSIS
Results

A total of 47 studies were included in the systematic review. Of these studies 66% involved only in vitro assessment, 6% involved only in vivo assessment, and 28% involved both in vitro and in vivo methods of assessment (Figure 1). Two general methods of preservation were utilized: culture (> 0°C) and cryopreservation (< 0°C) (Figure 2). Approximately 66% of studies used culture and 33% used cryopreservation (Figure 1).

Distribution of preservation methods (culture or cryopreservation; left) and assessment methods (in vivo and/or in vitro experimentation; right) of the 47 reviewed studies.

Two pie charts summarizing data from 47 studies. The left chart shows preservation methods: 66 percent used culture methods (31 studies) and 33 percent used cryopreservation (16 studies). The right chart shows assessment methods: 66 percent used in vitro only (31 studies), 6 percent used in vivo only (3 studies), and 28 percent used both in vitro and in vivo (13 studies). Islet culture

Islet culture studies were categorized by manipulation of temperature, oxygen conditions, media composition, use of scaffolds or alternative culture surfaces and co-culture. Most studies (21 of 31 studies) involved manipulation of a single factor (Table 4). Several other studies manipulated multiple factors (10 of 31 studies; Table 5).

Summary of studies, islet culture, single factor.

Studied parameter Study Method description Storage time, temperature Treatment groups Baseline viability Post-treatment viability Viability units Baseline GSIS Post-treatment GSIS GSIS conditions
Temperature Alcazar et al. 2020 (17) Compares duration of cold storage and duration of culture over a 24 h period 24 h8 or 37°C 1) 0 h at 8°C (24 h at 37°C)2) 22 h at 8°C (2 h at 37°C)3) 18 h at 8°C (6 h at 37°C)4) 6 h at 8°C (18 h at 37°C) Not reported Not reported Not reported Not reported 1) 3.002) 14.453) 7.364) 4.36 Low: 5.6 mMHigh: 16.7 mM
Oxygen Komatsu et al. 2016 (18) Compares culture oxygenation 7 days37°C 1) 21% O22) 50% O23) 35% O24) 10% O2 Not reported 1)150–250 µm: 91 ± 2*250–500 µm: 76 ± 4*2) 150–250 µm: 97 ± 0.5*250–500 µm: 91 ± 1*3) 150–250 µm: 95 ± 1*250–500 µm: 85 ± 3*4) 150–250 µm: 88 ± 2*250–500 µm: 55 ± 4* % live islet cells/total cells counted after fluorescein diacetate (FDA) and propidium iodide (PI) staining Not reported 1) 1.9 ± 0.22) 3.8 ± 0.53) 4.5 ± 0.74) 1.2 ± 0.2 Low: 3.3 mMHigh: 16.7 mM
Media Lee et al. 2008 (20) Compares media supplementation with human serum albumin (HSA) versus whole serum Overnight at 22°C + 48 h at 37°C 1) CMRL 1,066 + 0.5% HSA2) CMRL 1,066 + 10% serum Freshly isolated159 ± 21* 1) 103 ± 9*2) 80 ± 18* Islet equivalent (IEQ) Freshly isolated3.4 ± 0.8* 1) 2.4 ± 0.5*2) 1.9 ± 0.3* Low: 2 mMHigh: 16.7 mM
Nacher et al. 2016 (21) Compares media supplementation with human albumin versus ABO-compatible human serum 1 day37°C 1) CMRL 1,066 + 0.5% HSA2) CMRL 1,066 + 10% serum Not reported 1) 75.2 ± 4.52) 80.8 ± 4.4 % live islet cells/total cells counted after acridine orange (AO) and PI staining Not reported 1) 16 ± 5*2) 20 ± 4* Low: 2.8 mMHigh: 20 mM
3 days37°C 1) CMRL 1,066 + 0.5% HSA2) CMRL 1,066 + 10% serum Not reported 1) 75.3 ± 5.6%2) 91.7 ± 1.9% % live islet cells/total cells counted after AO and PI staining Not reported 1) 5 ± 0.5*2) 12.5 ± 2* Low: 2.8 mMHigh: 20 mM
Kerr-Conte et al. 2010 (22) Compares media supplementation with zinc, insulin, transferrin, selenium, in addition to AB serum (serum derived from donor blood of AB blood type) and Stem Ease, or linoleic acid, vitamin E and HSA 5 days37°C 1) Enriched CMRL 1,066 (CMRL 1,066 + zinc, insulin, transferrin, selenium)2) Enriched CMRL 1,066 + AB serum (2.5%) + Stem Ease3) Enriched CMRL 1,066 + linoleic acid + vitamin E + HSA (0.625%) 1) 90%*2) 97%*3) 90%* 1) 75%*2) 95%*3) 92%* % islets counted after culture/islets counted before culture 1) 3.7*2) 7.7*3) 5.0* 1) 2.0*2) 6.5*3) 4.6* Low: 2.8 mMHigh: 20 mM
Fraga et al. 1998 (23) Compares media supplementation with or without fetal bovine serum (FBS) 1 months37°C 1) CMRL 1,0662) CMRL 1,066 + 10% FBS Not reported 1) 79%*2) 57%* % live islet cells/total cells counted after dithizone staining Not reported 1) 2.7*2) 1.8* Low: 0 mMHigh: 20 mM
2 months37°C 1) CMRL 1,0662) CMRL 1,066 + 10% FBS 1) 65%*2) 46%* 1) 2.0*2) -
Ståhle et al. 2011 (24) Compares pathogen-inactivated, blood group compatible serum to nontreated human serum 3–4 days37°C 1) CMRL 1,066 + 10% serum2) CMRL 1,066 + 10% pathogen inactivated serum Not reported Not reported Not reported Not reported 1) 19.1 (median)2) 11.05 (median) Low: 1.67 mMHigh: 16.7 mM
Holmes et al. 1995 (25) Compares 10 different media for islet culture after 24 h in culture. The best performing media were selected for 7 days in culture and compared to RPMI 1,640 media control. 24 h37°C 1) RPMI 1,640 (11 mM glucose)2) RPMI 1,640 (2.2 mM glucose)3) Dulbecco's (25 mM glucose)4) Medium 199 (5.5 mM glucose)5) CMRL 1,066 (5.5 mM glucose)6) Iscove's (25 mM glucose)7) Waymouth's (27.7 mM glucose)8) Serum-free Serotec medium (25 mM glucose)9) Ex- cell 300 Serolab (20 mM glucose)10) Ham's F-12 (9 mM glucose) Not reported Not reported Not reported Not reported 1) 1.9*2) 2.0*3) 1.8*4) 2.2*5) 3.4*6) 2.3*7) 1.7*8) 1.5*9) 1.5*10) 2.4* Low: 1.7 mMHigh: 25.0 mM
7 days37°C 1) RPMI 1,640 (11 mM glucose)5) CMRL 1,066 (5.5 mM glucose)10) Ham's F-12 (9 mM glucose) Not reported Not reported Not reported Not reported 1) 2.0*5) 2.8*10) 1.5* Low: 1.7 mMHigh: 25.0 mM
Clayton et al. 2001 (26) Compares media supplementation with various concentrations of insulin 8 days37°C 1) CMRL 1,0662) CMRL 1,066 + 10 ng/ml insulin3) CMRL 1,066 + 100 ng/ml insulin4) CMRL 1,066 + 1,000 ng/ml insulin Not reported Not reported Not reported 1) 2.82 ± 1.292) 3.16 ± 2.043) 3.02 ± 1.184) 3.46 ± 1.47 1) 2.7 ± 1.382) 1.92 ± 0.373) 2.86 ± 0.94) 4.94 ± 5.39 Low: 2.8 mMHigh: 16.8 mM
Terra et al. 2011 (27) Assess the effect of culture with culture with recombinant human prolactin (rhPRL) after 24 h serum starvation 24 h starvation + 24 h culture37°C 1) CMRL 1,066 + vehicle2) CMRL 1,066 + rhPRL Not reported 1) 100%*2) 60%* % beta cells with fragmented nuclei/total beta cells (dead) Not reported Not reported Not applicable
24 h starvation + 48 h culture37°C 1) CMRL 1,066 + vehicle2) CMRL 1,066 + rhPRL Not reported 1) 100%*2) 55%* % of beta cells with fragmented nuclei/total beta cells (dead) Not reported Not reported Not applicable
Kaviani et al. 2019 (28) Compares the effects of culture with various concentrations of olesoxime 24 h37°C 1) CMRL 1,066 2) CMRL 1,066 + 0.1 uM olesoxime3) CMRL 1,066 + 1 uM olesoxime4) CMRL 1,066 + 10 uM olesoxime Not reported 1) 100%*2) 100%*3) 100%*4) 100%* % live islet cells/total cells counted after FDA and PI staining Not reported 1) 0.94 ± 0.1*2) 0.87 ± 0.2*3) 0.98 ± 0.1*4) 1 ± 0.2* Low: 2.8 mMHigh: 20 mM
72 h37°C 1) CMRL 1,066 2) CMRL 1,066 + 0.1 uM olesoxime3) CMRL 1,066 + 1 uM olesoxime4) CMRL 1,066 + 10 uM olesoxime Not reported 1) 95%*2) 95%*3) 97%*4) 97%* % live islet cells/total cells counted after FDA and PI staining Not reported 1) 0.5 ± 0.05*2) 0.26 ± 0.2*3) 0.7 ± 0.5*4) 1.8* Low: 2.8 mMHigh: 20 mM
Omori et al. 2010 (29) Compares the effects of culture with various concentrations of p38α-selective mitogen activated protein kinase inhibitor, SD-282 24 h37°C 1) CMRL 1,0662) CMRL 1,066 + DMSO3) CMRL 1,066 + 0.1 μM SD-282 (in DMSO)4) CMRL 1,066 + 0.3 μM SD-282 (in DMSO) Not reported Not reported Not reported Not reported 1) 2.9 ± 0.2*2) Not reported3) 4.7 ± 0.7*4) Not reported Low: 3 mMHigh: 16.8 mM
Media (Continued) Fornoni et al. 2008 (30) Assesses impact of c-jun N-terminal kinase (JNK) inhibition via supplementation with a small permeable TAT peptide JNK inhibitor known as L-JNKI Overnight37°C 1) Supplementation with control TAT peptide (10 μmol/L)2) Supplementation with L-JNKI peptide (10 μmol/L) 100% 1) 47.4 ± 8.2%2) 63.2 ± 12.8% % IEQ after culture/IEQ before culture after diphenylthiocarbazone staining Not reported Dynamic GSIR;No statistically significant differences were observed between C and 1 Low: 11 mMHigh: 25 mM
Bottino et al. 2002 (31) Compares media (CMRL 1,066 + 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mmol/L l-glutamine) without and with superoxide dismutases (SOD) mimic, AEOL10113 and AEOL10150 4 days37°C 1) Enriched CMRL 1,0662) CMRL 1,066 + SOD Mimic (34 μmol/L) 100% 1) 20% ± 5%*2) 21% ± 5%* % live islet cells/total cells counted after calcein-AM and PI staining Not reported 1) 5.5 ± 1.5*2) 5.8 ± 1.0* Low: 2.8 mMHigh: 20 mM
10 days37°C 1) Enriched CMRL 1,0662) CMRL 1,066 + SOD Mimic (34 μmol/L) 100% 1) 8% ± 5%*2) 14% ± 5%* % live islet cells/total cells counted after calcein-AM and PI staining Not reported Not reported Low: 2.8 mMHigh: 20 mM
Co-Culture de Souza et al. 2020 (40) Compares the effects of co-culture with adipose-derived stem cells (ASCs) 24 h37°C 1) w/o ASCs2) w/ indirect exposure to ASCs 92.3 ± 2.0% 1) 92 ± 2*2) 97 ± 1* % live islet cells/total cells counted after FDA and PI staining Not reported 1) 1.5 ± 0.25*2) 2.4 ± 0.3* Low: 2.8 mMHigh: 28 mM
48 h37°C 1) w/o ASCs2) w/ indirect exposure to ASCs 92.3 ± 2.0% 1) 91 ± 2*2) 96.5 ± 0.5* % live islet cells/total cells counted after FDA and PI staining Not reported 1) 1.4 ± 0.1*2) 2.6 ± 0.5* Low: 2.8 mMHigh: 28 mM
72 h37°C 1) w/o ASCs2) w/ indirect exposure to ASCs 92.3 ± 2.0% 1) 90.5 ± 2*2) 95.5 ± 1* % live islet cells/total cells counted after FDA and PI staining Not reported 1) 1.1 ± 0.3*2) ∼1.6* Low: 2.8 mMHigh: 28 mM
Surface/Scaffold Daoud et al. 2010 (71) Compares the effects of modifying the culture surface with various extracellular matrix components including collagen I, collagen IV, fibronectin, laminin, and bovine serum albumin (BSA) control 24 h37°C 1) BSA-modified surface2) Collagen I-modified surface3) Collagen IV-modified surface4) Fibronectin-modified surface5) Laminin-modified surface Not reported 1) 1.0*2) 0.8*3) 0.8*4) 0.95*5) 1.2* Cellular activity measured by WST-1 assay Not reported Not reported Not reported
48 h37°C 1) BSA-modified surface2) Collagen I-modified surface3) Collagen IV-modified surface4) Fibronectin-modified surface5) Laminin-modified surface Not reported 1) 1.0*2) 1.45*3) 1.1*4) 1.25*5) 1.0* Cellular activity measured by WST-1 assay Not reported Not reported Not reported
72 h37°C 1) BSA-modified surface2) Collagen I-modified surface3) Collagen IV-modified surface4) Fibronectin-modified surface5) Laminin-modified surface Not reported Not reported Not reported Freshly isolated 2.5* 1) 1.4*2) 1.0*3) 1.2*4) 1.4*5) 1.6* Low: 2.2 mMHigh: 22 mM
Maillard et al. 2011 (43) Compares the culture in fibrin, fibrin with non-emulsified perfluorodecalin (PDC) and fibrin with emulsified PDC 24 h37°C 1) No matrix2) Fibrin only3) Fibrin + non-emulsified PDC4) Fibrin + emulsified PDC Not reported 1) 81 ± 13%*2) 77 ± 13%*3) 76 ± 15%*4) 77 ± 16%* % live islet cells/total cells counted after FDA and ethidium bromide (EtBr) staining Not reported 1) 0.8*2) 0.7*3) 0.9*4) 1.4* Low: 2.75 mMHigh: 27.5 mM
Bentsi-Barnes et al. 2008 (45) Compares effects of islet culture on various gas-permeable membranes 48–90 h37°C 1) Nonadhesive tissue culture flask2) CS Hyde company cat no. 71-MED-DSP3) Bentec Medical cat no PR72034–04N4) Specialty Silicone Products cat no. SPM8234) Biorep Technologies Infusion Bag5) Baxter Lifecell Tissue Culture Bag cat no. R4R2111 >85% Not reported Not reported Not reported 1) 2.44 ± 0.582) 1.68 ± 0.473) 2.00 ± 0.394) 2.355) Extremely poor post-culture condition of the islets prevented evaluation6) 3.49 ± 0.64 Low: 3 mMHigh: 16.8 mM
Omori et al. 2024 (46) Compares outcomes of various durations of long-term storage in a poly-saccharide 3D-hydrogel (VitroGel 3D) within a gas permeable chamber 4 weeks37°C 1) Cell culture insert2) 3D scaffold Fresh Islets: 95% ± 1%* 1) 83% ± 2%*2) 92% ± 2%* % area of propidium iodide staining/area of Hoechst 33,342 staining Freshly isolated1.8 ± 0.1* 1) 3.4 ± 0.4*2) 3.4 ± 0.4* Low: 2.8 mMHigh: 28 mM
8 weeks37°C 1) 3D scaffold Fresh Islets: 93% ± 1%* 1) 92% ± 1%* % area of propidium iodide staining/area of Hoechst 33,342 staining Freshly isolated1.9 ± 0.3* 1) 2.3 ± 0.2* Low: 2.8 mMHigh: 28 mM
Woods et al. 2004 (47) Compares culture on porcine small intestinal submucosa (SIS) at varying time points. 5 weeks37°C 1) Cell culture insert2) Cell culture insert coated with SIS Not reported Not reported Not reported Not reported 1) 0.6 ± 0.6*2) 2.8 ± 0.7* Low: 4 mMHigh: 20 mM
Surface/Scaffold (Continued) Hadavi et al. 2019 (44) Compares the effects of cultures with various combinatorial ECM components with either poly(ester-urethane) (PEU) or poly(ethyleneglycol-terephthalatepolybutylene-terephthalate) (PEOT-PBT) microwell scaffolds relative to flat polystyrene (PS) plates. 3 days37°C Culture On PS Coated With:1a) Non-Coated1b) BSA1c) Fibronectin (FN) 1d) Collagen IV (Col4)1e) Laminin 111 (L111)1f) Laminin 332 (L332)1G) 20% FN:80% Col41H) 20% FN:80% L1111i) 20% FN:80% L3321j) 20% Col4:80% L1111k) 20% Col4:80% L3321l) 50% FN:50% Col41M) 50% FN:50% L1111n) 50% FN:50% L3321o) 50% Col4:50% L1111p) 50% Col4:50% L3321q) 80% FN:20% Col41r) 80% FN:20% L1111s) 80% FN:20% L3321t) 80% Col4:20% L1111u) 80% Col4:20% LN332 Culture on PEU coated with:2a) Non-coated2b) BSA2c) FN2d) Col42e) L1112f) L3322g) 20% FN:80% Col42h) 20% FN:80% L1112i) 20% FN:80% L3322j) 20% Col4:80% L1112k) 20% Col4:80% L3322l) 50% FN:50% Col42m) 50% FN:50% L1112n) 50% FN:50% L3322o) 50% Col4:50% L1112p) 50% Col4:50% L3322q) 80% FN:20% Col42r) 80% FN:20% L1112s) 80% FN:20% L3322t) 80% Col4:20% L1112u) 80% Col4:20% LN332 Culture on PEOT-PBT coated with:3a) Non-coated3b) BSA3c) FN3d) Col43e) L1113f) L3323g) 20% FN:80% Col43h) 20% FN:80% L1112i) 20% FN:80% L3323j) 20% Col4:80% L1113k) 20% Col4:80% L3323l) 50% FN:50% Col42m) 50% FN:50% L1113n) 50% FN:50% L3323o) 50% Col4:50% L1113p) 50% Col4:50% L3323q) 80% FN:20% Col43r) 80% FN:20% L1113s) 80% FN:20% L3323t) 80% Col4:20% L1113u) 80% Col4:20% LN332 Not reported Not reported Not reported Not reported 1a) 3.7*1b) 3.3*1c) 2.4*1d) 6.0*1e) 2.3*1f) 3.8*1g) 5.3*1h) 2.6*1i) 2.9*1j) 7.9*1k) 3.0*1l) 5.3*1m) 5.9*1n) 1.3*1o) 5.5*1p) 3.7*1q) 9.2*1r) 10.3*1s) 3.9*1t) 11.5*1u) 3.8* 2a) 4.4*2b) 4.7*2c) 4.3*2d) 8.6*2e) 2.4*2f) 5.4*2g) 6.8*2h) 4.3*2i) 7.8*2j) 4.5*2k) 6.4*2l) 3.8*2m) 4.0*2n) 1.1*2o) 4.5*2p) 4.2*2q) 3.3*2r) 1.8*2s) 1.82t) 5.7*2u) 3.1* 3a) 3.2*3b) 2.1*3c) 3.1*3d) 3.6*3e) 3.0*3f) 4.7*3g) 3.4*3h) 3.6*3i) 2.4*3j) 6.1*3k) 3.6*3l) 8.0*3m) 4.7*3n) 7.8*3o) 2.8*3p) 2.3*3q) 3.7*3r) 3.3*3s) 3.0*3t) 9.1*3u) 2.4* Low: 1.6 mmol/LHigh: 16.7 mmol/L
7 days37°C Culture on PS coated with:1a) Non-coated1b) BSA1c) FN1d) Col41e) L1111f) L3321g) 20% FN:80% Col41h) 20% FN:80% L1111i) 20% FN:80% L3321j) 20% Col4:80% L1111k) 20% Col4:80% L3321l) 50% FN:50% Col41m) 50% FN:50% L1111n) 50% FN:50% L3321o) 50% Col4:50% L1111p) 50% Col4:50% L3321q) 80% FN:20% Col41r) 80% FN:20% L1111s) 80% FN:20% L3321t) 80% Col4:20% L1111u) 80% Col4:20% LN332 Culture on PEU coated with:2a) Non-coated2b) BSA2c) FN2d) Col42e) L1112f) L3322g) 20% FN:80% Col42h) 20% FN:80% L1112i) 20% FN:80% L3322j) 20% Col4:80% L1112k) 20% Col4:80% L3322l) 50% FN:50% Col42m) 50% FN:50% L1112n) 50% FN:50% L3322o) 50% Col4:50% L1112p) 50% Col4:50% L3322q) 80% FN:20% Col42r) 80% FN:20% L1112s) 80% FN:20% L3322t) 80% Col4:20% L1112u) 80% Col4:20% LN332 Culture on PEOT-PBT coated with:3a) Non-coated3b) BSA3c) FN3d) Col43e) L1113f) L3323g) 20% FN:80% Col43h) 20% FN:80% L1112i) 20% FN:80% L3323j) 20% Col4:80% L1113k) 20% Col4:80% L3323l) 50% FN:50% Col42m) 50% FN:50% L1113n) 50% FN:50% L3323o) 50% Col4:50% L1113p) 50% Col4:50% L3323q) 80% FN:20% Col43r) 80% FN:20% L1113s) 80% FN:20% L3323t) 80% Col4:20% L1113u) 80% Col4:20% LN332 Not reported Not reported Not reported Not reported 1a) 2.9*1b) 2.1*1c) 5.3*1d) 3.9*1e) 3.7*1f) 7.6*1g) 4.7*1h) 3.8*1i) 2.5*1j) 4.7*1k) 6.6*1l) 4.2*1m) 3.1*1n) 5.3*1o) 3.0*1p) 4.0*1q) 3.1*1r) 2.7*1s) 2.0*1t) 9.3*1u) 3.5* 2a) 6.8*2b) 5.0*2c) 5.1*2d) 6.0*2e) 4.7*2f) 1.3*2g) 2.0*2h) 3.6*2i) 2.0*2j) 2.9*2k) 3.4*2l) 7.8*2m) 4.6*2n) 2.0*2o) 7.9*2p) 3.1*2q) 1.8*2r) 3.6*2s) 1.6*2t) 16.3*2u) 1.6* 3a) 4.1*3b) 3.6*3c) 3.5*3d) 5.0*3e) 3.8*3f) 1.8*3g) 3.0*3h) 3.0*3i) 3.0*3j) 12.7*3k) 2.8*3l) 2.9*3m) 4.4*3n) 3.0*3o) 3.3*3p) 2.1*3q) 2.3*3r) 4.0*3s) 1.2*3t) 15.0*3u) 3.3* Low: 1.6 mmol/LHigh: 16.7 mmol/L

Denotes values that were not directly reported by the study authors but instead extracted from the published figures.

Summary of studies, islet culture, multiple factors.

Studied parameter Study Method description Storage time, temperature Treatment groups Baseline viability Post-treatment viability Viability units Baseline GSIS Post-treatment GSIS GSIS conditions
Temperature + Oxygen Komatsu et al. 2019 (19) Compares culture at various temperature and oxygen culture conditions 2 weeks12, 22, or 37°C 1) 37°C with 21% O22) 12°C with 21% O23) 12°C with 50% O24) 22°C with 21% O25) 22°C with 50% O26) 37°C with 50% O2 100% 1) 56% ± 2%2) 82% ± 3%3) 92% ± 2%4) 79% ± 1%5) 85% ± 1%6) 65% ± 2% % islet volume post-culture/islet volume pre-culture Freshly isolated1.85 ± 0.2 2) 1.9 ± 0.2 Low: 2.8 mMHigh: 28 mM
Temperature + Media Noguchi et al. 2010 (32) Compares culture at various temperatures and using various solutions 48 h4, 22, or 37°C 1) CMRL 1,066 + 0.5% HSA Miami #1 at 37°C2) CMRL 1,066 + 0.5% HSA Miami #1 at 22°C3) University of Wisconsin (UW) solution at 4°C 2,000 IEQ 1) 1,525 ± 29 IEQ2) 1,621 ± 26 IEQ3) 1,900 IEQ IEQ Not reported Not reported Low: 2.8 mMHigh: 25 mM
Jay et al. 2004 (33) Compares culture and preservation at various temperatures and using various solutions 18 h at in the test conditions directly after isolation4, 22–24, or 30°C 1) TCM199 30°C2) TCM199 22°C3) UW 4°C4) Eurocollins solution 4°C Not reported 1) 0.223 ± 0.1582) 0.201 ± 0.1593) 0.611 ± 0.9924) 0.205 ± 0.123 ATP/ADP ratio Not reported 1) 2.41 ± 1.132) 1.76 ± 1.083) 1.19 ± 0.304) 1.14 ± 0.29 Low: 2 mMHigh: 15 mM
Overnight culture, then 4 h in the test conditions4, 22–24, or 30°C 1) TCM199 at 30°C2) TCM199 at 22°C3) UW solution at 4°C4) Eurocollins solution at 4°C Not reported 1) 0.199 ± 0.0692) 0.178 ± 0.0553) 0.173 ± 0.0854) 0.137 ± 0.018 ATP/ADP ratio Not reported 1) 2.12 ± 0.582) 1.73 ± 0.513) 1.36 ± 0.344) 2.07 ± 0.63 Low: 2 mMHigh: 15 mM
Shindo et al. 2022 (34) Compares various culture medias and preservation solutions at various temperatures 48 h4, 22, or 37°C 1) CMRL at 4°C2) CMRL at 22°C3) CMRL at 37°C4) CMRL at 37°C for 24 h, then at 22°C for 24 h5) PRODO at 4°C6) PRODO at 22°C7) PRODO at 37°C8) PRODO at 37°C for 24 h, then at 22°C for 24 h9) UW at 4°C Not reported 1) 94% ± 5%*2) Not reported3) Not reported4) Not reported5) Not reported6) 98% ± 1%*7) 98% ± 1%*8) 99% ± 1%*9) 98% ± 1%* % live islet cells/total cells counted after FDA and PI staining Freshly isolated islets: 6.0 ± 4.0 1) 1 ± 0.75*2) Not reported3) Not reported4) Not reported5) Not reported6) 3 ± 1*7) 6.5 ± 4*8) 4 ± 1.5*9) 1 ±  0.5* Low: 1.67 mMHigh: 16.7 mM
Delfino et al. 1993 (10) Compares various cold culture solutions 6 days4°C 1) Hanks’ balanced salt solution2) UW3) Sumimoto D4) Histidine-lactobionate 1) 152) 14.23) 154) 15 1) 4.22) 9.03) 7.54) 7.5 Viability score after FDA and EB staining where a score of 15 represents a fully viable islet Not reported Not reported Not reported
Rush et al. 2004 (36) Compares effects of extended culture between 1 and 6 months in Memphis serum-free media (M-SFM) composed of Connaught Medical Research Laboratories (CMRL) 1,066 with HEPES, ZnSO4, and NaOH 1 months28°C 1) M-SFM 100% 1) 86.67 ± 1.53 % IEQ after culture/IEQ before culture Not reported 1) 2.15 ± 0.28 Low: 60 mg/dlHigh: 300 mg/dl
3 months28°C 1) M-SFM 100% 1) 58.33 ± 18.45 % IEQ after culture/IEQ before culture Not reported 1) 2.4 ± 1.74 Low: 60 mg/dlHigh: 300 mg/dl
6 months28°C 1) M-SFM 100% 1) 39.67 ± 12.58 % IEQ after culture/IEQ before culture Not reported 1) 1.18 ± 0.46 Low: 60 mg/dlHigh: 300 mg/dl
Oxygen + Media Brandhorst et al. 2017 (38) Compare the effects of hypoxic (2% O2) culture in preconditioned Minimum Essential Media α (MEMα) supplemented with Glutamax, 10% FCS and getamycin. The media was preconditioned via mesenchymal stem cell (MSC) culture under normoxic (21% O2) or hypoxic (1% O2) conditions for 2 days. 3–4 days37°C 1) MEMα, 2% O22) MEMα preconditioned via 21% O2 MSC culture, 2% O23) MEMα preconditioned via 1% O2 MSC culture, 2% O2 Not reported 1) 59 ± 22) 59 ± 33) 61 ± 3 % live islet cells/total cells counted after FDA and PI staining Not reported 1) 1.0  ± 0.12) 1.4  ± 0.13) 1.4  ± 0.1 Low: 2 mMHigh: 20 mM
Lemaire et al. 2023 (37) Compares the effects of supplementing media with two marine worm hemoglobins, M101 and M201, in hypoxic conditions. Oxygen is manipulated by varying islet seeding density and oxygen tension 24 h37°C 1) 150 IEQ/cm² in CMRL1,066 with 21% O22) 600 IEQ/cm² in CMRL1,066 with 21% O2 Not reported 1) 85 ± 6%*2) 87 ± 4%* % live islet cells/total cells counted after FDA and PI staining Not reported 1) 4.2 ± 0.2*2) 3.0 ± 0.5* Low: 2.8 mMHigh: 16.7 mM
24 h37°C 1) CMRL1,066, 21% O22) CMRL1,066 with M101, 21% O23) CMRL1,066 with M201, 21% O24) CMRL1,066, 2% O25) CMRL1,066 with M101, 2% O26) CMRL1,066 with M201, 2% O2 Not reported 1) 84 ± 3*2) 93 ± 1* 3) 94 ± 1*Not reported for 2% O2 % live islet cells/total cells counted after FDA and PI staining Not reported 1) 2.0 ± 0.2*2) 3.1 ± 0.4*3) 2.2 ± 0.5*4) Not reported5) Not reported6) 2.8 ± 0.5*
Media + Surface/Scaffold Lucas-Clerc et al. 1993 (72) Compares the effect of media [minimum essential medium (MEM) + 5.5 mM glucose or RPMI + 11 mM glucose] and culture surface (on culture-treated plastic, within collagen gel, or on top of collagen gel) 25 days37°C 1) MEM on plastic2) MEM on collagen3) MEM in collagen4) RPMI on plastic5) RPMI on collagen6) RPMI in collagen Not reported Not reported Not reported 1) 6.20 ± 0.4* 1) No secretion2) 1.9 ± 0.3*3) 1.5 ± 0.2*4) No secretion*5) 2.4 ± 0.3*6) 1.6 ± 0.2* Low: 2.75 mMHigh: 22 mM
Co-Culture + Mechanical Stimulation Murray et al. 2009 (41) Compares individual culture or co-culture with pancreatic ductal epithelial cells under static or rotational culture conditions 10 days37°C 1) Static culture2) Static culture w/ epithelial cells3) Rotational culture4) Rotational culture w/ epithelial cells Not reported Not reported Not reported Not reported 1) 1.2*2) 1.5*3) 1.2*4) 1.8* Low: 1.67 mMHigh: 16.7 mM
Temperature

Cold cell culture has been associated with prolonged cell viability, as metabolic processes slow down, thereby reducing protein degradation. Alcazar et al. 2020 focused their investigation on the duration of cold culture (8°C) over a 24-hour period and the resulting effects on islet function (17). A longer cold storage period was associated with a higher dynamic GSIS index.

 Oxygen

Another critical factor for islet viability and function is oxygenation. Komatsu et al. 2016 studied varied oxygen tensions (10%, 21%, 35%, 50%) over a 7-day culture period at 37°C, concluding that hyperoxia (35%, 50%) helps maintain islet volume and GSIS (18). A further study builds on this work by investigating the combined effects of optimizing temperature and oxygen conditions in islet cultures. Via a 2-week islet culture, Komatsu et al. 2019 explored several temperatures (12°C, 22°C, 37°C) combined with oxygenation adjustments (21%, 50%) on a 2-week culture (19). The most effective combination, 12°C with 50% oxygenation, was not statistically significantly different from freshly isolated islets in terms of viability or GSIS (19).

 Media composition

Twelve studies investigated islet culture medium composition alone. An additional 9 studies focused on the impact of media in combination with another factor, such as temperature, oxygen or scaffold.

Connaught Medical Research Laboratories 1,066 medium (CMRL 1,066) has been widely used in pre-transplantation islet culture studies due to its ability to inhibit β-cell depolarization, preserve cellular function, and enhance glucose responsiveness (7). Lee et al. 2008 and Nacher et al. 2016 both compared CMRL 1,066 islet culture media supplemented with 10% human serum vs. 0.5% human albumin (20, 21). While both groups cultured the human islets for 3 days at 37°C, these studies provided conflicting evidence. Lee et al. 2008 concluded that albumin is superior to human serum (20), while Nacher et al. 2016 reported that human serum more effectively preserves islet viability and GSIS (21). Kerr-Conte et al. reported that 2.5% human serum was superior to 0.625% albumin for both 1 and 5 day culture (22). For long-term storage, Fraga et al. 1998 found that serum-free islet culture led to better viability and function as compared to culture supplemented with 10% FBS (23). Ståhle et al. found that pathogen-inactivation of serum did not influence islet outcomes. Discrepancies between the investigations may have resulted from differences in other conditions, such as culture temperature, in addition to methodology for assessing islet viability and GSIS (24).

Insulin and glucose concentrations in culture also affect islet function. Holmes et al. 1995 cultured islets for 1 week in media formulations with various glucose concentrations ranging from 2.2 to 27.7 mM (25). Holmes and colleagues found that CMRL 1,066 supplemented with 5 mM (90 mg/dl) glucose yields the highest GSIS after both 24 hours and 7 days in culture (25). Variability between isolations prevented Clayton et al. 2001 from making conclusions regarding the effects of insulin concentration in culture medium on islet viability and function (26).

Other studies utilized media additives that have been shown to mitigate cellular apoptosis [e.g., human recombinant prolactin (rhPRL), olesoxime] (27, 28), inhibit proinflammatory cytokine production [e.g., p38α-selective mitogen activated protein kinase inhibitor SD-282 (29), c-Jun N-terminal kinase inhibitor L-JNKI (30)], or break down toxic superoxide radicals [e.g., superoxide dismutase (SOD) mimics] (31).

Five studies combine alterations in temperature and media. A commonality among many of the studies was to assess culture in various mediums at 22°C and 37°C and compare to cold culture in various organ preservation solutions at 4°C (32–35). There was not a consensus regarding the optimal temperature for islet preservation. For 4°C storage, all four studies showed that University of Wisconsin (UW) solution, commonly used for solid organ flushing and cold storage, was associated with the best outcomes. Other studies fixed temperature and assessed alternative solutions. For example, Rush et al. 2004 cultured islets in serum-free media at 28°C for 6 months and demonstrated marginal viability and function (36).

A single study assessed both oxygen and media supplementation (37). Marine worm hemoglobins M101 and M201 were evaluated as a supplement to human islet culture at normoxic and hypoxic conditions due to its associated anti-inflammatory and antioxidant properties. Moreover, these hemoglobins were investigated as oxygen carriers due to their high oxygen-binding capacity, which may help mitigate the hypoxic conditions commonly encountered during pre-transplant islet storage. Oxygen conditions were manipulated either by modifying islet seeding density or oxygen tension. In both normoxic and hypoxic conditions, the marine worm hemoglobin improved islet viability and glucose stimulation index (GSI)—a ratio reflecting insulin secretion at high vs. low glucose derived from the GSIS assay—compared to islets cultured in unsupplemented media.

Brandhorst et al. 2017 cultured islets under hypoxic conditions (2% oxygen) in mesenchymal stem cell (MSC) preconditioned medium under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions (38). MSCs are multipotent stromal cells derived from connective tissues, with immunomodulatory and regenerative properties, including the secretion of anti-inflammatory proteins and growth factors that may prevent β-cell apoptosis and support islet cell survival and function (39). The preconditioned media improved GSI relative to the control. No difference in GSI was observed between the preconditioned media from MSCs cultured under normoxic or hypoxic conditions.

 Co-culture

Additionally, co-culturing islets with other cell types has shown promise in enhancing islet health and reducing cellular stress. Stem cells or epithelial cells have been reported to generate a supportive microenvironment for islets (38, 40, 41). After 72 hour culture, islets cocultured with indirect contact to adipose-derived stem cells were 95.2 ± 1% viable with GSIS of 1.6 compared to viability 90.5 ± 2% with GSIS 1.1 ± 0.3 without coculture (40). While pancreatic ductal cell co-culture had some preservative effect on islet GSIS after 10 days in culture relative to islets cultured alone, significance was only observed when cultured in a rotational system (41).

 Culture surfaces and scaffolds

Seven studies utilized modified culture surfaces or scaffolds in efforts to improve viability by enhancing engraftment and oxygen delivery. Most of these studies (4 of 6) focused on creating culture surfaces that mimic the native extracellular matrix (ECM). Daoud et al. 2010 and Maillard et al. 2011 assessed ECM-component scaffolds and fibrin matrices with perfluorodecalin (PDC) (42, 43). Daoud's study utilized a poly(lactide-co-glycolide) acid (PGLA) scaffold embedded with collagen I gel, fibronectin, and collagen IV. By optimizing pore size, after 10 days in culture, islets showed GSIS on par with freshly isolated islets (42). Maillard's work found that fibrin with emulsified PDC decreased hypoxia and improved GSIS after 24 hours in culture (43).

Hadavi et al. 2019 found that functionalization of a scaffold with ECM components was more important than the choice of material for the scaffold. Both Hadavi et al. 2019 and Daoud et al. 2011 found that displaying a combination of ECM components (as compared to a single component) was critical to preserve islet viability and function long term (42, 44).

Two studies focused on investigating gas-permeable membranes as alternatives to a traditional culture flask (45, 46). Bentsi-Barnes et al. 2008 investigated a variety of commercial membranes and found that after 48 hours of culture, the Baxter Lifecell Tissue culture bag most effectively preserved GSIS (45). When cultured on other gas-permeable membrane products, islets did not survive or showed functional decline inferior to non-adherent tissue culture flasks (45). Omori et al. 2024 found that human islets cultured on poly-saccharide 3D-hydrogel (VitroGel 3D) within a gas permeable chamber had enhanced viability after 4 weeks in culture, but no difference in GSI compared to islets cultured in suspension (46).

In contrast, Woods et al. 2004 explored using porcine small intestinal submucosa as a substrate for functional islet recovery (47). After 5 weeks in culture, islets on small intestinal submucosa had a GSI of 2.8 ± 0.7 compared to 0.6 ± 0.6 for control islets.

Early experimentation by Lucas-Clerc et al. 1993 assessed both culture surface and media composition. Islets cultured on plastic were compared to those cultured in or on collagen gel. Additionally, MEM + 5.5 mM glucose was compared to Roswell Park Memorial Institute 1640 Medium (RPMI) + 11 mM glucose. RPMI is rich in amino acids, vitamins, glucose, salts, and a bicarbonate buffer that are biochemically necessary for cell survival. After 17 days in culture, islets cultured on plastic had no secretion response to glucose stimulation, while those cultured in or on collagen gel retained some responsiveness (GSI: 1.50–2.40). Islets cultured on collagen retained function in a superior manner (GSI: 1.90–2.40) to those cultured in the collagen (GSI: 1.50–1.60). RPMI + 11 mM glucose (GSI: 1.60–2.40) was found to be superior to MEM + 5.5 mM glucose (GSI: 1.50–1.90) for both islets cultured in and on collagen (40).

A comprehensive summary of all reviewed papers on islet culture is provided in Table 4 (Single Factor) and 4 (Multiple Factors).

 Cryopreservation

Cryopreservation is a promising alternative strategy for islet preservation, in which cells are frozen to −196°C in order to arrest cellular metabolism. When frozen, water no longer solvates solutes, creating an increasingly concentrated solution that causes cell injury via osmotic dehydration (48). Cryoprotectant selection is critical to mitigating damage to islets during the cryopreservation process. Cryoprotectant prevents ice crystal formation from damaging cells by permeabilizing the cell membrane. However, cell membrane permeabilization can also be toxic, impairing functional recovery. Herein, 13 studies utilizing cryopreservation to preserve islets were analyzed (Tables 6, 7). While islet (1–3 months) culture outcomes are superior at early timepoints (49), Misler et al. 2005 found that islets could be preserved via cryopreservation using dimethyl sulfoxide (DMSO) for 2 years. After 1 or 2 days of recovery in culture, insulin secretion and single-cell action potential were not statistically significantly different from fresh islets (50).

Summary of studies, islet cryopreservation, single factor.

Studied parameter Study Method description Cooling method Thawing methods Storage time Treatment groups Baseline viability POST-treatment viability Viability units Baseline GSIS Post-treatment GSIS GSIS conditions
Culture vs. Cryopreservation Misler et al. 2005 (50) Compares cryopreservation using 2.0 M DMSO to fresh isolation Slow cooling (0.25°C/min) to −40°CStorage at −196°C Rapid warming (200°C/min) with cytoprotectant dilution with sucrose 2 years storage1–2 days recovery in culture before assessment 1) Cryopreservation Not reported Not reported Not reported Freshly isolated 7.5 ± 1.5* 1) 5.8 ± 1.2* Low: 3 mMHigh: 15 mM
Gaber et al. 2001 (49) Compares serum-free culture versus cryopreservation Slow cooling (0.25°C/min) to −40°CStorage at −70°C Not reported 2 months 1) Culture2) Cryopreservation Not reported Not reported Not reported Short-term culture (2–5 days)5 ± 3.35 1) 3.31 ± 1.522) 3.18 ± 2.19 Low: 60 mg/dlHigh: 300 mg/dl
Vitrification Langer et al. 1999 (56) Compares culture, cryopreservation and vitrification Subcooled to −7.2°C, slow cooling (0.25°C/min), to −40°CStorage at −196°C Rapid warming (200°C/min) with cytoprotectant dilution with sucrose, and stepwise dilution with isotonic medium Not reported 1) Culture2) Cryopreservation3) Vitrification Freshly isolated85.6  ± 1.4% 1) Not reported2) 51.8  ± 3.0%3) 17.3 ± 8.0% % live islet cells/total cells counted after FDA and PI staining Freshly isolated 13.9 1) 13.92) 6.13) Not reported Low: 30 mg/dlHigh: 300 mg/dl
Jutte et al. 1987 (57) Compares culture and vitrification at various timepoints post-isolation using vitrification media containing 0% vitrification medium consists of 0.3% bovine serum albumin, 20.5% DMSO, 15.5% acetamide,10% propylene glycol and 4.5% polyethylene glycol (MW: 6,000) Stepwise cooling to 0°C with stepwise cryoprotectant concentrationStorage at −196°C Rapid warming (200°C/min) with stepwise cytoprotectant dilution Not reportedImmediate assessment 1) Culture, 6 days2) Culture, 10–13 days3) Vitrification 2 days after isolation4) Vitrification 6–9 days after isolation Not reported 1) 97% ± 2%2) 100% ± 0%3) 80 ± 8%4) 85 ± 3% % islets counted after treatment/ islets counted before treatment of islets counted before treatment Not reported Not reported Not reported
Not reported4 days recovery in culture before assessment 1) Culture, 6 days2) Culture, 10–13 days3) Vitrification 2 days after isolation4) Vitrification 6–9 days after isolation Not reported 1) 97% ± 2%2) 100% ± 0%3) 88 ± 6%4) 94 ± 2% % islets counted after treatment/ islets counted before treatment of islets counted before treatment Not reported 1) 2.25*2) 2.29*3) 1.89*4) 1.88* Low: 2.5 mMHigh: 25 mM
Cryoprotectant Lakey et al. 2001 (51) Compares various concentrations of cytoprotectants DMSO or ethylene glycol (EG), and various addition methods (stepwise or one-step) Slow cooling (0.25°C/min) to −40°CStorage at −196°C Rapid warming (200°C/min) with cytoprotectant dilution with sucrose 1 week storage2 days recovery in culture before assessment 1) Cryopreservation, 2.0 M DMSO, stepwise2) Cryopreservation, 1.5 M DMSO, stepwise3) Cryopreservation, 1.5 M DMSO, one-step4) Cryopreservation, 2.0 M EG, stepwise5) Cryopreservation, 1.5 M EG, stepwise6) Cryopreservation, 1.5 M EG, one-step 100% 1) 62% ± 4%*2) 74% ± 3%*3) 69% ± 3%*4) 52% ± 4%*5) 64% ± 5%*6) 51% ± 7%* % islet volume post-culture/islet volume pre-cryopreservation Not reported 1) 4.5 ± 0.5*2) 6.0 ± 0.4*3) 6.5 ± 0.8*4) 3.8 ± 0.5*5) 3.2 ± 0.4*6) 3.5 ± 0.5* Low: 2.8 mMHigh: 20 mM
Kojayan et al. 2019 Compares different concentrations of cytoprotectants DMSO and EG Slow cooling (0.25°C/min) to −40°CStorage at −196°C Rapid warming (200°C/min) with cytoprotectant dilution with sucrose 4 weeks storage2 days recovery in culture before assessment 1) Cryopreservation, 2 M DMSO2) Cryopreservation, 1 M DMSO + 1 M EG3) Cryopreservation, 1 M DMSO + 0.5 M EG 1) 92%2) 92%3) 92% 1) 52 ± 3%*2) 78 ± 2%*3) 80 ± 2%* % live islet cells/total cells counted after FDA and PI staining 1) 3.5*2) 3.5*3) 3.5* 1) 2.1 ± 0.4*2) 3.2 ± 0.2*3) 3.4 ± 0.4* Low: 2.8 mMHigh: 28 mM
Omori et al. 2007 (54) Compares cryopreservation using an intracellular-ion islet cryopreservation solution (ICS) without or with a p38 MAPK inhibitor (SD-282/p38IH; ICS-p38IH) Slow cooling (0.3°C/min) to −50°CStorage at −196°C Rapid warming with cytoprotectant with sucrose Not reportedImmediate assessment 1) Cryopreservation, RMPI, 2.1 M DMSO2) Cryopreservation, ICS, 2.1 M DMSO3) Cryopreservation, ICS, 2.1 M DMSO + p38IH 91% ± 4%* 1) 89% ± 4%*2) 92% ± 3%*3) 92% ± 1%* % live islet cells/total cells counted after FDA and PI staining Not reported Not reported Not reported
Not reported2 days recovery in culture before assessment 1) Cryopreservation, RPMI2) Cryopreservation, ICS3) Cryopreservation, ICS-p38IH 91% ± 4%* 1) 86% ± 3%*2) 87% ± 2%*3) 88% ± 3%* % live islet cells/total cells counted after FDA and PI staining 4.1 ± 0.6* 1) 1.8 ± 0.2*2) 2.0 ± 0.3*3) 2.6 ± 0.2* Low: 3 mMHigh: 19 mM
Kenmochi et al. 2008 (53) Assessment of hydroxyethyl starch (HES) to reduce DMSO toxicity. Cooled with a programmed freezing system, Cryomed Model 1,010 Rapid warming in a 37°C water bath and resuspended with RPMI-1,640 containing 10% FBS 2 weeks–3 months storage1 h recovery in culture before assessment 1) Cryopreservation, RPMI 1,640 with 5% DMSO, 6% HES, and 4% FBS 80,349 ± 37,164 1) 57,595 ± 31,027 IEQ 3.37 ± 3.02 1) 1.34 ± 0.28 Low: 3.3 mMHigh: 20 mM
Recovery Protocols Komatsu et al. 2017 (61) Compares thawing and recovery in culture after cryopreservation under high atmospheric oxygen environments Storage at −196°C Rapid thawing in 37°C water bath with stepwise cytoprotectant dilution with sucrose 3 months storage2 days recovery in culture before assessment 1) 50% O2 Thaw: 50% O2 Culture2) 50% O2 Thaw: 21% O2 Culture3) 21% O2 Thaw: 50% O2 Culture4) 21% O2 Thaw: 21% O2 Culture 1) 95.8%2) 95.8%3) 96.2%4) 96.2% 1) 78% ± 6%*2) 67% ± 3%*3) 66% ± 3%*4) 62% ± 3%* % islet volume post-thaw/islet volume pre-cryopreservation Not reported 1) 2.8 ± 0.4*2) 2.6 ± 0.1*3) 2.3 ± 0.4*4) 2.0 ± 0.3* Low: 3.3 mMHigh: 16.7 mM
Kneteman et al. 1989 (58) Compares allowing DMSO to equilibrate for 15 min at 0°C or 0°C before cryopreservation Supercooled to −7.5°C, slow cooling (0.25°C/min) to −40°CStorage at −196°C Rapid warming (200°C/min) to 25°C or 0°C with cytoprotectant dilution with sucrose 46 days storageImmediate assessment 1) Cryopreservation, DMSO equilibration at 0°C2) Cryopreservation, DMSO equilibration at 25°C Not reported 1) 94.2 ± 3.5%2) 95.0 ± 8.9% % islet volume post-thaw/islet volume pre-cryopreservation Not reported Not reported Not reported
46 days storage24 h recovery in culture before assessment 1) Cryopreservation, DMSO equilibration at 0°C2) Cryopreservation, DMSO equilibration at 25°C Not reported Not reported Not reported 7.7 ± 1.8 1) 4.3 ± 1.02) 3.7 ± 1.2 Low: 60 mg/dlHigh: 300 mg/dlGlucose perfusion peak/basal SI
46 days storage48 h recovery in culture before assessment 1) Cryopreservation, DMSO equilibration at 0°C2) Cryopreservation, DMSO equilibration at 25°C Not reported Not reported Not reported 7.7 ± 1.8 1) 6.2 ± 0.82) 6.0 ± 1.2 Low: 60 mg/dlHigh: 300 mg/dlGlucose perfusion peak/basal SI
Beattie et al. 1997 (60) Compares cryoprotectant dilution with standard sucrose or trehalose during rapid rewarming Supercooled to 7.5°C, slow cooling (0.25°C/min) to −40°CStorage at −196°C Rapid warming with cytoprotectant dilution with sucrose or trehalose Unspecified 1) Cryopreservation, cryoprotectant dilution with 750 mM sucrose2) Cryopreservation, cryoprotectant dilution with 300 mM trehalose 100% 1) 58%2) 92% % total DNA extracted from recovered islets/total DNA extracted from fresh islets 2.08 1) 2.462) 2.48 Low: 1.6 mMHigh: 16.7 mM
Janjic et al. 1996 (59) Assess the effects of the presence of the antioxidants butylated hydroxyanisole (BHA) and vitamin K1 during thawing and recovery in culture Slow cooling from −4°C to −40°C (0.3°C/min), then −40°C to −170°C (5°C/min) Cryotubes incubated in 37°C water bath 24–36 h storage3 h recovery in culture before assessment 1) Cryopreservation2) Cryopreservation, BHA (100 μM)3) Cryopreservation, Vitamin K1 (5 μg/ml) Not reported Not reported Not reported Not reported 1) 1.35*2) 2.46*3) 2.00* Low: 2.8 mMHigh: 16.7 mM

Summary of studies, islet cryopreservation, multiple factors.

Study Method description Cooling method Thawing methods Storage time Treatment groups Baseline viability Post-treatment viability Viability units Baseline GSIS Post-treatment GSIS GSIS conditions
Zhan et al. 2022 (62) Compares vitrification and rewarming on a nylon (38-um pore size) cryomesh with an optimized cryoprotectant agent formulation of 22% EG and 22% DMSO to conventional cryopreservation technique using 0.5 M EG + 1 M DMSO or 2 M DMSO Cryopreservation: Slow cooling (0.25°C/min) to −40°CVitrification: Vitrification (∼59,600°C/min) Cryopreservation: 200°C/minVitrification: ∼280,000°C/min 9 m storage 1) Cryopreservation2) Vitrification Freshly isolated 92.3%Ethanol killed 2% 1) 59.1–62.2%2) 87.4% % live islet cells/total cells counted after AO and PI staining Freshly isolated 4.5 ± 2.0 1) 3.75 ± 1.252) 3.65 ± 1.50 Low: 3.3 mMHigh: 16.7 mM

Many studies have compared various concentrations of cryoprotectants DMSO and ethylene glycol (EG). Work by Lakey et al. 2001 compared various concentrations (1.5 M and 2.0 M) of DMSO and EG, added to the culture in a stepwise manner or all at once. DMSO yielded greater islet post-thaw recovery as compared to EG. 1.5 M DMSO yielded superior post-cryopreservation viability and GSIS as compared with 2.0 M treatment. No significant difference was observed between stepwise and one-step addition (51). Kojayan et al. 2019 compared 2 M DMSO alone and 1M DMSO plus 0.5 or 1M EG. Results indicated that 1 M DMSO with 0.5 M EG was the most effective (52). Kenmochi et al. 2008 found that the addition of hydroxyethyl starch (HES) could be used to reduce the required concentration of DMSO, thereby reducing associated toxicity (53). Of note, no controls assessments were used in Kenmochi's study.

In addition to combatting cellular damage from ice crystal formation, supplements have been used to inhibit inflammatory processes. Omori et al. 2007 found that supplementation of an intercellular cryopreservation solution with p38 inhibitor SD-282 enhanced post-storage GSIS relative to conventional medium or intracellular during islet cryopreservation (54).

Vitrification

Vitrification is a type of cryopreservation in which freezing occurs more quickly, preventing ice crystals from forming. Vitrification requires direct plunge of cells treated with vitrification solution into −196°C liquid nitrogen. Theoretically, supercooling of the cryoprotective solution solidifies it into a metastable, highly viscous glass phase that limits ice formation, molecular diffusion, and metabolic activity. To achieve vitrification rapid cooling and rewarming occur at a rates of approximately −200°C/min and 250°C/min respectively (55). However, in the studies reviewed herein, vitrification failed to result in superior outcomes with respect to islet viability or function post-storage (56, 57).

 Thawing

In addition to the freezing process, islet thawing can also impact islet viability. Kneteman et al. 1989 studied the impact of the rewarming temperature after DMSO cryopreservation (58). Islets were rapidly warmed to 0°C or 25°C. However, no significant difference was observed between the treatment groups. A few years later, Janjic et al. 1996 and Beattie et al. 1997 reported that the addition of agents that combat DMSO toxicity during rewarming improved outcomes for islets (59, 60). Janjic and coauthors demonstrated that the addition of antioxidants butylated hydroxyanisole (BHA) or vitamin K1 during thawing and recovery improved GSI. Beattie et al. showed that substituting the sucrose in cryoprotectant dilution solution with trehalose improved islet viability as measured via extracted DNA, however no difference was observed in GSI (60). Komatsu et al. 2017 exposed islets to high atmospheric oxygen during the thawing process. GSIS was found to be the highest in the treatment group that received the highest oxygen concentration during thawing (50%) and culture (50%) (61).

Zhan et al. optimized many of the previously discussed factors impacting cryopreservation (62). This group used vitrification to both quickly freeze and thaw islets on a nylon cryomesh in an optimized cryopreservation solution consisting of 22% DMSO and 22% EG. The optimized techniques enabled islet storage for 9 months with minimal reduction in viability and GSI.

 <italic>In vivo</italic> experiments

Of the 47 studies included in this systematic review, 13 conducted additional in vivo experiments following in vitro work, while 3 other studies involved only in vivo testing. Seven studies utilized culture storage techniques (Table 8), and 9 studies utilized cryopreservation (Table 9). All these in vivo experiments involved transplanting stored human islets into the renal subcapsular space in an animal model. Immunocompromised mice were used in all studies, except for one, in which immunocompetent C57BL/6 mice were used (56). Most studies utilized nonobese diabetic-severe combined immunodeficiency (NOD-scid). Other studies used Rag1, BALB/C nude, NMRI nude, or athymic nude-Foxn1nu. Two studies reported the use of nude mice without further clarification (32, 63).

Summary of studies, in vivo, culture.

Study Mouse strain Diabetes induction IEQ transplanted Transplantation site Treatment groups Storage time Outcomes Xenograft results description
Bottino et al. 2002 (31) NOD-scidRag 1 Streptozotocin (STZ) 200–1,000 IEQ Kidney capsule 1) Culture, Enriched CMRL 1,0662) Culture, CMRL 1,066 + SOD Mimic (34 μmol/L) 2 h Normoglycemia SOD mimic significantly improved outcomes1) With 700–1,000 IEQ, restored normoglycemia in 100% of mice within 10 days. With 200 or 400 IEQ, restored normoglycemia in 50% and 80% of mice, respectively2) Regardless of transplanted IEQ, restored normoglycemia in 100% of mice within 10 days
Noguchi et al. 2010 (32) Nude STZ 2,000 IEQ Kidney capsule C) Freshly isolated1) Culture, CMRL 1,066 + 0.5% HSA Miami #1 at 37°C2) Culture, CMRL 1,066 + 0.5% HSA Miami #1 at 22°C3) Culture, UW solution at 4°C 48 h Normoglycemia C) Restored normoglycemia in 86.7% of mice (13/15)1) Restored normoglycemia in 15.4% of mice (2/13)2) Restored normoglycemia in 50% of mice (3/6)3) Restored normoglycemia in 53.3% of mice (8/15)
Nacher et al. 2016 (21) Athymic nude-Foxn1nu STZ 2,000 IEQ Kidney capsule 1) Culture, CMRL 1,066 + 0.5% HSA2) Culture, CMRL 1,066 + 10% Serum 3 days Normoglycemia No significant difference was observed over 60 days.
Omori et al. 2024 (46) NOD-scid STZ 1,200 IEQ Kidney capsule C) Freshly isolated1) Culture, 3D scaffold 4 weeks NormoglycemiaImmunofluorescent staining for insulin, glucagon and somatostatin C) Restored normoglycemia in 66.7% of mice (8/14)1) Restored normoglycemia in 71.4% of mice (5/7)
Rush et al. 2004 (36) NOD-scid STZ 250, 500,1,000 or 2,000 IEQ Kidney capsule 1) Culture, M-SFM 1, 3 or 6 months NormoglycemiaHuman insulinHuman C-peptide M-SFM cultures of up to 6 months can improve outcomes for both 1,000 and 2,000 IEQ implantations1) Restored normoglycemia in 100% of 1,000 IEQ and 2,000 IEQ transplanted mice% (5/5 and 5/5) with optimal insulin and C-peptide levels up to 3 months and reduced but functional levels at 6 months
Komatsu et al. 2019 (19) NOD-scid STZ 1,200 IEQ Kidney capsule C1) Freshly isolated, PIM-RC1) Freshly isolated, CMRL 1,0661) Culture, PIM-R, 12°C, 50% O₂2) Culture, CMRL 1,066, 12°C, 50% O₂ 2 weeks NormoglycemiaHistology No significant difference in restoration of normoglycemia or histology was observed.C1) Restored normoglycemia in 75% of mice (6/8)C2) Restored normoglycemia in 80% of mice (8/10)1) Restored normoglycemia in 75% of mice (6/8)2) Restored normoglycemia in 78% of mice (7/9)
Chen et al. 2019 (73) NOD-scid STZ 200 or 400 hand-picked islets Kidney capsule 1) Culture, transwell2) Culture, transwell + nanofibrillar cellulose (NFC) hydrogel 31 days NormoglycemiaHuman C-peptide NFC hydrogel significantly improved outcomes.1) Failed to restore normoglycemia in any mice2) Mean blood glucose reached normoglycemia from day 14 to 28 before rising, with C-peptide levels peaking on day 8 at 109.6 ± 33.8 pmol/L and persisting through day 18
Ståhle et al. 2011 (24) NMRI nude Alloxan 3,000 IEQ Kidney capsule 1) Culture, CMRL 1,066 + 10% serum2) Culture, CMRL 1,066 + 10% pathogen inactivated serum 3–4 days Normoglycemia No significant difference was observed.1) Restored normoglycemia in 87% of mice (8/9)2) Restored normoglycemia in 78% of mice (7/9)
Omori et al. 2010 (29) NOD-scid STZ 1,200 IEQ Kidney capsule 1) Culture, CMRL 1,0662) Culture, CMRL 1,066 + 0.1 μM SD-282 (in DMSO) 24 h NormoglycemiaGlucose tolerance test SD-282 significantly improved outcomes1) Restored normoglycemia in 25% of mice (1/4)2) Restored normoglycemia in 100% of mice (5/5); Had significantly better responses to glucose challenge compared with control
Fornoni et al. 2008 (30) Athymic nude-Foxn1nu STZ 500, 1,000, or 2,000 IEQ Kidney capsule 1) Culture, TAT peptide only2) Culture, L-JNKI treated 48 h NormoglycemiaGlucose tolerance test Despite no significant improvement, L-JNKI treated islets displayed improved glucose tolerance from days 16–120 and similar normoglycemia rates1) With 1,000 IEQ, restored normoglycemia in 75% of mice (3/4)2) With 1,000 IEQ, restored normoglycemia in 100% of mice (5/5)

Summary of studies, in vivo, cryopreservation.

Study Mouse strain Diabetes induction IEQ transplanted Transplantation site Treatment groups Storage time Outcomes Xenograft results description
Ricordi, et al. 1988 (74) Balb/c nude STZ 400–600 islets Kidney capsule 1) Cryopreservation 2–8 weeks NormoglycemiaHistology: Aldehyde Fuchsin, H&E Duration of study: 45 days1) Within 3 weeks, restored normoglycemia in 100% of mice (4/4); Histology showed viable, revascularized islets
Kneteman et al. 1989 (58) Balb/c nude No induction 200 islets Kidney capsule 1) Cryopreservation 46.5 days (median) Histology: insulin Duration of study: 14 days1) Immunohistochemistry confirms intact islet granules within the renal subcapsular space in 87.5% of mice (7/8)
Piemonti et al. 1999 (63) Nude STZ 1,000 hand-picked islets Kidney capsule C) Freshly isolated1) Cryopreservation 5–30 days NormoglycemiaGlucose tolerance test No significant difference in survival was observed.Duration of study: 240 daysC) Surviving mice maintained vivo function at 90 d as indicated by IVGTT1) Surviving mice failed to maintain in vivo function at and after 90 d as indicated by IVGTT
Langer et al. 1999 (56) C57BL/6 No induction 1,000 IEQ Kidney capsule C) Freshly isolated1) Cryopreservation Not reported Insulin recovery No significant difference was observed.C) 25.6  ± 7.3% insulin recovery after transplant1) 24.1  ± 7.4% insulin recovery after transplant
Omori et al. 2007 (54) NOD-scid STZ 1,600 IEQ Kidney capsule C) Freshly isolated1) Cryopreservation with RPMI2) Cryopreservation with ICS3) Cryopreservation with ICS-p38IH 60 Normoglycemia Duration of study: 90 daysDiabetic mice were implanted with an insulin pellet for the first 2 weeks following transplant.C) Restored normoglycemia in 85.7% of mice (6/7)1) Became hyperglycemic when insulin implant was removed2) Became hyperglycemic when insulin implant was removed3) Restored normoglycemia in 80% of mice (4/5)
No induction 1,000 IEQ Kidney capsule C) Freshly isolated1) Cryopreservation with ICS2) Cryopreservation with ICS-p38IH 60 Human C-peptide Duration of study: 32 daysNo human C-peptide was detected in nondiabetic mice transplanted with human islets for at least 3 weeks post-transplant. After 3 weeks, C-peptide was detected:C) Secreted the highest concentration of C-peptide1) Secreted minimal C-peptide2) Increased to 86% of the C-peptide level of the freshly isolated islet group (C)
Gaber et al. 2001 (49) NOD-scid No induction 2,000–3,000 IEQ Kidney capsule 1) Culture2) Cryopreservation 60 days Human C-peptide No significant difference was observed.Duration of study: 126 days

In most studies, the rodents were rendered diabetic via chemical induction with streptozotocin or alloxan. In 3 studies, diabetes was not induced (49, 56, 58). Between 200 and 3000 IEQ were transplanted. 10 studies involved cultured islets, and 6 studies involved cryopreservation.

In all studies, islets were transplanted to the kidney capsule. Stored islets reversed diabetes in animal models at similar rates to fresh islets in most studies, although islet equivalents were often equal despite greater loss of viable islets in the long-term storage treatment groups. For transplantation studies, the reported measurements varied greatly between studies. Studies reported oral glucose tolerance tests, C-peptide levels, and blood glucose levels at various timepoints and frequencies. Endpoints for sacrifice and islet morphological analysis ranged from 14 days post-transplantation to up to 126 days.

 Discussion

Experimentation with human islet storage, both via culture and cryopreservation, shows promising results for a future where islets can be banked for effective islet transplantation in as many patients as possible. Lowering culture temperatures, increasing oxygenation, and utilizing ECM-component scaffolds can all improve the viability and function of islets in culture. For cryopreservation, optimization of cryoprotectant concentrations and oxygenation while thawing can reduce islet loss. Culture and cryopreservation supplementation offer further mitigation of the stress-induced damage that islet cells incur.

Study limitations include the heterogeneity of results and methods reported in the reviewed studies. The National Institutes of Health Clinical Islet Transplantation (NIH CIT) consortium established a standard operating procedure for glucose stimulated insulin secretion in 2014 with low glucose concentrations of 2.8 mM and high glucose concentrations of 28 mM (64). Many studies occurred before publication of this SOP and its widespread implementation. While GSIS was a ubiquitous measure of islet function used in the studies reviewed, low and high glucose concentrations used varied widely.

Since the focus of this systematic review was cryopreservation and culture techniques with clinical applicability, the study population was limited to human islets. Many studies relevant in terms of topic were not relevant in terms of population. Human islet preservation remains relatively underexplored compared to experimentation with islet models derived from animals. Advances in scaffolding and reaggregation of cryopreserved human islets with the Insphero 3D InSight Islet Biology Platform may accelerate the study of human islet preservation (65).

This study was limited to cryopreservation and did not explore high subzero methods of preservation such as supercooling, partial freezing, and isochoric subzero. Studies in solid organ preservation using high subzero techniques have shown promise in human liver and rat liver and heart models (66, 67). Another promising approach to addressing the limited supply of freshly isolated human islets that was not explored in this review is utilization of human stem cell derived islets. These clinical trials have investigated the efficacy and safety of autologous and allogeneic mesenchymal stem cell derived islet-like organoids for type 1 and type 2 diabetes therapy (68). Wang et al.'s transplantation of chemically induced pluripotent stem cells into the anterior abdominal rectus sheath of a Type 1 Diabetic patient on preexisting immunosuppression for a liver transplant showed sustained insulin independence, lowered HbA1C, and improved glucose response to oral glucose tolerance test 1-year post transplantation (69). Recently, the VX-880-101 FORWARD study of zimislecel, Vertex Pharmaceuticals’ allogeneic stem cell-derived islet-cell therapy, published promising phase 1–2 study results (70). While the study size is small (n = 14), long-term follow up shows significant sustained decreases in HbA1C, total daily insulin dose, and time out of target glucose range (70–180 mg/dl) (70). At day 365, 10 of 12 participants achieved insulin independence (70).

Zhan et al.'s cryopreservation study highlights that optimizing multiple factors is essential to achieving long-term islet viability and function (62). Success in this complex field also demands a multidisciplinary approach and diverse expertise. Optimization of cryopreservation parameters of human islets remains a relatively underexplored field compared to that of human islet culture. Most studies in this systematic review report on the results of cryopreservation alone or compare cryopreservation to similar length cultures. Extending the possible lifespan of freshly isolated islets is a new opportunity. The ability to stockpile islets for “off the shelf” transplantation would greatly improve the treatment options for patients, especially those outside of Chicago, where Lantidra treatment is currently available. As the market for Lantidra grows, cryopreserved human islets’ impact upon FDA approval will also grow.

 Author contributions

AC: Methodology, Data curation, Writing – review & editing, Investigation, Conceptualization, Writing – original draft, Formal analysis. JC: Writing – original draft, Writing – review & editing, Investigation, Visualization, Data curation, Formal analysis, Validation. JB: Writing – review & editing, Funding acquisition, Writing – original draft, Visualization, Conceptualization, Supervision, Investigation.

 Funding

The author(s) declare that financial support was received for the research and/or publication of this article. The study was supported by The Northwestern Summer Research Program for Medical Students (NIH NIDDK T35 5T35DK126628-04), in addition to a Strategic Research Agreement (3-SRA-2023-1389-S-B) and Diversifying Diabetes Research Talent in Academia Award (2-SRA-2023-1452-S-B) from BreakthroughT1D (formerly JDRF).

 Acknowledgments

This is a short text to acknowledge the contributions of specific colleagues, institutions, or agencies that aided the efforts of the authors.

 Conflict of interest

JB has financial interests in SNC Therapeutics, Inc.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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