This study was conducted following the methodological guidance provided by the Joanna Briggs Institute for scoping reviews and is reported in accordance with the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) checklist, which promotes transparency and rigor in scoping review reporting (Peters et al., 2020; Tricco et al., 2018).

Eligibility criteria were established as a priori to include original research articles in the review. Articles were screened by three investigators based on their titles and abstracts. Next, the full texts of all the records that contained terms related to human HCC were collected and uploaded into Zotero version 7.0.3 (https://www.zotero.org/). The methodology of each was independently reviewed by two investigators. Articles that were included (1) directly compared hPL with FBS in HCC (2) involved culture in plastic-only (e.g., not coated/treated), containers, and (3) were written in English. Articles were excluded if they (1) did not include an FBS control, (2) declared previous contamination with FBS before culture in the alternative, and (3) used a different basal media for the alternative and FBS. Discrepancies between the findings of the two investigators were resolved by consulting with the third investigator.

A structured search was performed, by querying three electronic databases: PubMed, Google Scholar, and the FCS-free database from 1 January to 30 July 2024. The relevant MeSH terms and entry terms were based on the PICO (population, intervention, comparison, and outcome)-structured question: “Could human platelet lysate be applied as an FBS substitute?”, where, P: human HCC; I: human platelet lysate (hPL); C: FBS; and O: HCC performance characteristics, including fold expansion, viability, identity, and functionality.

To query PubMed and Google Scholar, using the search syntax, “(Hematopoietic Stem Cells OR Hematopoietic Cells) AND (Cell Culture Techniques OR Cell Cultures) AND (Fetal Bovine Serum OR FBS OR Fetal Calf Serum OR FCS) AND (Serum-Free Media OR Xeno-Free Media OR Xeno-Free Supplement) AND (Human Platelet Lysate OR hPL OR Platelet Lysate OR Human Serum OR AB Serum OR Pooled Plasma) AND (Cell Proliferation OR Cell Expansion OR Cell Viability)” resulted in several hits including other cell types. Consequently, the search was broadened by removing “(Hematopoietic Stem Cells OR Hematopoietic Cells) AND.” To retrieve articles from the FCS-free database (https://fcs-free.sites.uu.nl/), the “literature” and “mammalian” filters were selected under source and species, respectively. A collection of records was compiled using Microsoft Excel and duplicate data was not included.

Two investigators performed data extraction independently. For the included studies, relevant data were extracted from the main text, tables, and figures, as well as supplementary files. The following data were collected for each study: (1) hPL production method; (2) supplement concentration; (3) cell morphology; (4) cell viability; (5) fold expansion data; (6) cell identity; and (7) functional assays.

Variables in the hPL production protocols were identified across studies. The cytokine regimes, basal media, and other additives were also collected, along with the concentration of hPL or FBS supplementation. The following parameters were qualitatively analyzed across the included studies: fold expansion, cell viability, identity evaluation, and functional assays.

As shown in Figure 1A, the initial search identified 622 relevant records. Nine records were removed before screening, including duplicates and non-English language texts. Another 558 records were excluded based on screening their titles and abstracts. The full texts of 55 records were analyzed and 50 did not satisfy one or more pre-defined criteria. Finally, five studies that compared hPL and FBS groups were included in this review. Data were extracted from two main areas: hPL production method as well as HCC conditions and performance indicators (Figure 1B).

As it provides information on cell health and behavior, cell morphology is an important phenotypic indicator of HCC performance. However, only two studies compared the effects of hPL and FBS supplementation on cell morphology using inverted light microscopy (Švajger, 2017; Jabbarpour et al., 2023). Švajger (2017) examined the morphology of dendritic cells (DCs) after differentiation from peripheral blood mononuclear cells (PBMCs; Day 6) and activation into other subtypes (Day 8). On Day 6, circular monocytes became ‘potato-like’ during their differentiation to immature DCs (iDCs) in both FBS and hPL supplemented cultures, with no observable morphological differences. Additionally, no morphological differences were observed after the activation of iDCs to mature dendritic cells (mDCs) and tolerogenic dendritic cells (TolDCS) using lipopolysaccharide and 1,25-dihydroxyvitamin D3, respectively. Thus, this study reported equal efficacy in maintaining the morphology of DC subtypes using FBS and hPL at 10% supplementation.

Meanwhile, Jabbarpour et al. (2023) reported observable morphological differences between FBS and hPL supplemented cytokine-induced killer (CIK) cells, after differentiation from PBMCs (Day 15). Three supplement concentrations were tested (2.5, 5, and 10%). Compared to 10%, fewer aggregates were observed at lower concentrations in both FBS and hPL, supporting that supplement concentration directly affects cell proliferation. However, aggregates were notably denser and irregular in size and shape across all three concentrations of FBS compared to hPL. The differences between FBS and hPL were most pronounced at 10% supplementation, as FBS resulted in far fewer aggregates, which were larger and denser. hPL supplemented aggregates were more uniform, smaller and greater in number. A similar trend was observed at lower concentrations. These results suggest that hPL might support more physiologically relevant culture conditions, as denser clusters in FBS supplementation may promote the acquisition of culture-induced mutations and provide a competitive advantage for these clones.

While both Švajger (2017) and Jabbarpour et al. (2023) explored the effects of FBS and hPL on cell morphology, their findings highlight important nuances in supplement performance. Švajger (2017) reported no significant morphological differences between FBS and hPL in DC development, indicating comparable support for immune cell phenotypes. In contrast, Jabbarpour et al. (2023) observed distinct morphological disparities in CIK cells, with hPL producing more uniform and less dense aggregates than FBS. These findings imply that hPL may better preserve normal cell behavior and morphology, potentially reducing the risk of culture-induced anomalies. Together, the two studies that examine morphological differences between FBS and hPL supplementation point towards the possibility of morphological differences based on supplement concentration, as well as hematopoietic cell type.

Fold expansion is the ratio between the final number of cells and the initial number of seeded cells. It is an important consideration for scaling HCC to clinically relevant volumes in applications where the total cell yield is the primary concern. The differentiation of PBMCs and CD34⁺ cells into desired cell types requires careful consideration of several key factors, including basal media, cytokine regime, additives, and stimulants. Table 1 summarizes these factors across the included studies. However, only three out of the five studies compared fold expansion in FBS and hPL supplementation. Two studies compared the expansion of lymphoid cells from PBMCs and one study compared fold expansion of myeloid cells from UCB-derived CD34⁺ cells.

In myeloid cells, Yaghoubi et al. (2021) reported a small increase in the fold expansion at 10% supplementation using in-house hPL compared to FBS in CD34⁺ cells; however, this difference was not significant. Jabbarpour et al. (2023) reported a higher fold expansion of T-cells using in-house hPL compared to FBS across all three concentrations that were tested (2.5, 5, and 10%). Meanwhile, at 5% supplementation, commercially produced hPL decreased fold expansion compared to FBS (Canestrari et al., 2019). While there is limited data to enable meaningful comparison, the methods implemented to produce commercial hPL compared to in-house drastically differ and may account for the variability in the results obtained across studies.

Although only one study reporting fold expansion also performed biochemical profiling of hPL, it is reasonable to assume that growth factor concentrations can vary depending on the method of hPL production. Canestrari et al. (2019) found relatively low levels of IGF-1 (∼100 pg/mL) and TGF-β1 (150–200 pg/mL) across ten batches of commercially produced hPL. In contrast, Zamani et al. (2021) reported substantially higher concentrations of IGF-1 (111.1 ± 53.37 ng/mL) and TGF-β (27.18 ± 13.19 ng/mL) across five batches of in-house hPL, although they did not measure fold expansion in monoculture. It is worth noting that the values reported in in-house hPL are in range to reported levels of IGF-1 and TGF-β in FBS, which are 46 ± 1.6 ng/mL and 55 ± 0.6 ng/mL, respectively (Mohamed et al., 2020). ELISA was used to measure growth factor concentrations in all of these studies. Additionally, Canestrari et al. (2019) reported that commercial hPL was abundant in proteins involved in eliciting inflammatory and immune responses, which may suppress cell proliferation and contribute to lower fold expansion. The use of cryopreserved rather than freshly isolated PBMCs may further affect these results. Therefore, a comprehensive evaluation of hPL and FBS efficacy must consider production methods, growth factor profiles, and the source of PBMCs.

Cell viability, a measure of the proportion of live and healthy cells in a population, is an important indicator of cell culture performance. To compare the cell viability in hPL and FBS, annexin-V and 7-Aminoactinomycin D as well as trypan blue staining methods were used in the included studies.

Using double negative staining for annexin-V and 7-Aminoactinomycin D, Švajger (2017) measured cell viability at the end of the differentiation period (Day 6) of DCs that were cultured from PBMCs. Double negative staining is advantageous over other methods as it is accurate and sensitive, allowing the allowing distinction between live, early, late apoptotic/necrotic cells. Annexin-V is known to bind to phosphatidylserine in apoptotic cells as 7-Aminoactinomycin D stains the DNA in dead/late apoptotic cells, which have permeabilized membranes. Using flow cytometry, viability can be evaluated with more sensitivity and accuracy compared to other methods. The results demonstrated a 5% lower cell viability in hPL compared to FBS, but the results were not significant and showed more than 70% viability in both conditions.

Other staining methods reported higher viability in hPL and FBS supplemented cells. Trypan blue exclusion assay is a semi-quantitative method commonly used to obtain rough estimates of live versus dead cells in culture. Using Trypan blue exclusion assay, the viability of T-cells after differentiation in the presence of IL-2 from cryopreserved PBMCs (Day 14) was preserved (>90%) in both hPL and FBS, with no significant difference between the two conditions (Canestrari et al., 2019). Jabbarpour et al. (2023) reported a similar range, although it is unclear what method was used to obtain these results. Nevertheless, significantly higher viability was reported in hPL than FBS across three concentrations (2.5, 5, and 10%) at all time points. The viability in FBS was concentration-dependent, as lower viability was observed at concentration <10%, the least being at 2.5%. Meanwhile, viability remained >90% across all concentrations of hPL.

There is a lot of variability in the results regarding cell viability between the two supplementation groups, which may be attributed to hPL production method as well as method used to evaluate viability. In-house hPL presented the most variability ranging from 70% to >90%. However, similar results observed in FBS cultures, indicating that assay type may account for reported variability across studies. These results underscore the potential to replace hPL as an effective alternative for maintaining healthy cell populations in HCC but highlight the requirement for better reporting standards across cell culture experiments.

One of the challenges in HCC is the spontaneous commitment to differentiated phenotypes, which makes it difficult to obtain relevant volumes of desired cell types. Hematopoietic cells can be differentiated by their unique cell surface protein profiles. Using flow cytometry, phenotypic prevalence and expression level of markers can provide information on the functional and differentiation status of cultured cells. Best practices include reporting both parameters; however, in studies where yield was a priority, only phenotypic prevalence was reported. On the other hand, studies where immunophenotypic changes during differentiation were important, expression levels of markers were presented. Table 2 summarizes the findings of the studies that reported phenotypic prevalence across hPL and FBS groups. Figure 2 illustrates the various cell types examined across the included studies. Although cell morphology, fold expansion, and viability results varied drastically across studies, hPL supplementation had a positive effect on maintaining cell identity across all the cell types that were evaluated in the included studies.

Švajger (2017) demonstrated that by Day 6, hPL supplementation effectively supported the generation of iDCs with high expression of characteristic markers, evaluated by flow cytometry. Expression of monocyte-associated markers (low CD14 and CD16; high CD1a and DC-SIGN) was comparable between FBS- and hPL-supplemented cultures, although CD1a expression was notably reduced with hPL. The DC-associated markers CD11b and CD11c were expressed at higher levels in FBS conditions. Evaluation of HLA molecules revealed similar HLA-I expression levels between groups, while HLA-II expressions were elevated in hPL cultures. Following activation with lipopolysaccharide (20 ng/mL), the expression of co-stimulatory molecules CD40 and CD83 was lower in hPL-supplemented cells, whereas CD80 and CD86 levels remained comparable. Notably, the lymph node-homing receptor CCR7 and HLA-II, indicative of DC maturation, were slightly higher in hPL conditions. Furthermore, upon treatment with 1,24-dihydroxyvitamin D₃, ILT-3 expression, associated with tolerogenic potential, increased by two-fold in hPL cultures compared to FBS. Collectively, these findings suggest that hPL is a suitable alternative to FBS for DC differentiation and may offer advantages in promoting a TolDC phenotype.

Canestrari et al. (2019) reported that compared to FBS supplementation, hPL has a capacity to produce T cells of less differentiated subtypes. In this study, T-cell maturation was measured after CD3/CD28 stimulation by immunostaining for CD62L or a combination of CCR7 and CD45RO to assess CD4⁺ and CD8⁺ T-cell subtypes. As shown in Table 2, hPL consistently showed a significantly increased proportion of cells expressing CCR7 and CD45RO, indicative of the central memory phenotype (T_CM). Additionally, an increased percentage of CD62L⁺ cells was observed, further supporting the enrichment of both naïve and T_CM cells in hPL cultures. These findings suggest that hPL not only supports T cell expansion but also promotes a more desirable, less terminally differentiated phenotype, which may be advantageous in adoptive immunotherapy applications.

Yaghoubi et al. (2021) evaluated megakaryocyte lineage markers from UCB-derived CD34⁺ cells using flow cytometry on Day 8. Significantly higher proportion of cells expressed CD41 marker in hPL compared to FBS supplementation. A slight increase in the proportion of cells expressing CD42b in hPL was also observed; however, the difference was not significant. CD41 and CD42b are megakaryocyte markers, and the increase observed in hPL compared to FBS supplementation, potentially suggest enhancement of proplatelet formation. Similarly, Zamani et al. (2021) reported an increase in the percentage of cells expressing early to intermediate erythroid progenitor markers, CD71 and glycophorin A, in hPL compared to FBS on Day 7. These findings suggest that hPL not only supports lineage-specific differentiation but also promotes the maintenance of more desirable, less terminally differentiated progenitor cell types compared to FBS, which may enhance both the efficiency and functional potential of HCC.

Jabbarpour et al. (2023) evaluated cell identity of CIK cells across three supplement concentrations (2.5, 5, and 10%). Archetypal cell marker of CIK cells reflects their hybrid T/natural killer-like phenotype. At lower concentrations, the proportion of cells expressing CIK markers, CD3 and CD56, were lower than at 10% in both cultures. However, at 5% supplementation, the proportion of cells expressing these markers were double that of FBS (Table 2). T-cell marker expression (CD3⁺CD56⁻) was comparable between both cultures and across all three concentrations (>95%). Canestrari et al. (2019) reported similar results as no significant difference was observed in the proportion of T-cells (CD3⁺CD56⁻), although the percentage of cells was slightly higher in hPL than FBS. These results suggest that hPL is an effective alternative to FBS in obtaining differentiated phenotypes of lymphoid lineages.

Overall, the studies demonstrated that hPL is a viable and, in many cases, advantageous alternative to FBS in maintaining the identity and desirable phenotypes of hematopoietic-derived cells. hPL supplementation supported the expression of lineage-specific surface markers of DC, T cells, megakaryocytes, erythroid progenitors, and CIK cells. Moreover, hPL allowed the retention of less terminally differentiated states, including TolDCs, T cells, and early-stage erythroid and megakaryocytic progenitors. Together, these findings highlight hPL’s potential to enhance both the functional quality and translational relevance of HCC for clinical applications.

Pooling strategies and solutions also vary widely across studies, contributing to differences in hPL quality and batch-to-batch variability. Zamani et al. (2021) reported high batch-to-batch variability in the levels of growth factors measured across five batches, which pooled five PCs each. Canestrari et al. (2019) suggest that using at least 100 donations per batch may overcome biological variation arising from donors. Viau et al. (2019) reported that across 14 batches, pooling 200 donations in additive solution, containing 25%–40% residual plasma, reduced the coefficient of variance (CV) <9%. Whereas using only five donations had a greater CV of 18%–30%. Conversely, Fazzina et al. (2015) demonstrated that pooling 12 PC donations in ∼600 mL of InterSol solution (Fenwall Inc.) and 20%–30% human plasma showed no relevant variation in the growth factor concentration across 20 batches. More recently, Bianchetti et al. (2021) recommended pooling 25 PC donations in cryoprecipitated plasma reduced CV <9%. However, Zamani et al. (2021) reported high batch-to-batch variability using 25 donations. Therefore, a systematic study that evaluates the effect of pooling strategies and solutions is needed to standardize hPL production methods.

Most studies used a simple and cost-effective method of repeated freeze-thaw cycles to break down platelets for hPL preparation. Specifically, three out of five HCC studies used a three-cycle protocol at −80°C for freezing and +37°C for thawing. One study found success with just a single freeze-thaw cycle at −80°C and +2°C–8°C (Švajger, 2017). Additionally, Canestrari et al. (2019) used electron beam (E-beam) irradiation to lyse the cells. No other study implemented pathogen reduction treatments. There was little evidence to suggest that one platelet lysis method may provide a better hPL biochemical profile than another. Instead, the choice of methods implemented by different groups was based on cost-effectiveness and convenience on handling volumes. Bianchetti et al. (2021) found that lower cycle counts increased variability in growth factor release, while four cycles achieved optimal stability and growth factor consistency. Similarly, across MSC studies, doubling time was lower in hPL produced from at least three cycles (Palombella et al., 2022). Therefore, standardizing protocols to perform three to four freeze-thaw cycles for platelet lysis could reduce variability and enhance hPL’s effectiveness in HCC.

Plasma contains a variety of clotting factors, including fibrinogen and prothrombin. Prothrombin is converted to thrombin, which produces fibrin from fibrinogen. Thus, fibrin depletion or control is recommended when using hPL to prevent the gelation of culture media. Hemeda et al. (2013) reported that the addition of heparin was critical to maintaining low-glucose, DMEM in a liquid state for MSC culture. The hPL used in this study was made from PCs, which were prepared by suspending 2.0–4.2 × 10¹¹ platelets in 200 mL plasma containing acid-citrate-dextrose (ACD). PCs were subjected to two freeze-thaw cycles. However, heparin can reduce cell viability, alter cell behavior, and significantly affect miRNA expression (Hemeda et al., 2014; Ling et al., 2016; Oeller et al., 2024).

Heparin is commonly obtained from porcine or bovine mucosal tissue, which can complicate clinical applications and introduce variability in hPL-supplemented cell culture experiments. To develop a completely xeno-free culture, Mojica-Henshaw et al. (2013) investigated the feasibility of converting hPL-plasma to hPL-serum for MSC culture. hPL-plasma was obtained by subjecting PC (1.0 × 10⁹ platelets/mL) to a freeze-thaw cycle. For hPL-serum, CaCl₂ was added to PC and allowed to clot overnight. The clot was removed, and the supernatant was collected. In MSCs cultured in α-MEM, both hPL-plasma and serum resulted in similar immunophenotype profile and tri-lineage differentiation; however, hPL-plasma resulted in higher cell yields compared to hPL-serum. Thus, the need for heparin may depend on the hPL production method, which could leave the fibrinogen and coagulation factors intact.

Typically, the fibrinogen levels reported in hPL produced using freeze-thaw cycles, sonication, and solvent-detergent chemical treatment ranged from 1.5 to 3.0 g/L, which requires the addition of 0.6 to 2.0 IU/mL of heparin in the cell culture media (Chou and Burnouf, 2016). Meanwhile, methods including serum conversion and removal of clotting factors through the addition of CaCl₂ may not require heparin addition. In a study that directly compared the two methods, fibrinogen was present in freeze-thawed hPL, but no thrombin proteolytic activity was detected (Delila et al., 2021). On the other hand, fibrinogen was <0.4 mg/mL in serum-converted hPL (using 23 mM of CaCl₂) and high thrombin proteolytic activity was observed. The activity significantly reduced in heated serum-converted hPL, which may suggest that heat might affect thrombin activity.

A recent systematic review evaluating the effectiveness of hPL to replace FBS in MSC, reported that four out of the 29 included studies did not add heparin during hPL production nor to cell culture (Palombella et al., 2022). The absence of heparin did not affect the doubling time. Mohamed et al. (2020) reported that cultures with Ca content <0.25 mM could not induce clot and did not require heparin. In this study, no gelation was observed in RPMI-1640 (0.4 mM of Ca) whereas heparin at a minimum concentration of 0.25 IU/mL was required for DMEM (1.5 mM of Ca) cultures. Two of the five studies that were included in this review used RPMI-1640 for HCC and one study added 2 IU/mL (Jabbarpour et al., 2023). Meanwhile two other studies used IMDM (1.5 mM of Ca) and did not add heparin or implement any other measures. AIM-V was used as a basal media in one study, and the composition of this media is not publicly available.

Given the variability in the application of fibrinogen depletion across studies, greater transparency and consistency across studies could be achieved by reporting key parameters that enable comparison. These should include the fibrinogen concentration and thrombin proteolytic activity of the hPL, as well as the final Ca concentration in the culture medium. Standardized reporting of these factors would help clarify their impact on experimental results and support reproducibility across research settings.

Although most of the hPL production process is performed in a closed loop, and the starting material undergoes sterility checks according to national and international blood bank requirements, PCs are susceptible to pathogen contamination. While risks are lower in hPL produced using small pooling strategies, contamination from a single donation can have disastrous effects in larger pooling strategies (>16 donations). However, the selection of pathogen reduction method and frequency may affect hPL performance in cell culture (Bieback et al., 2019). Studies confirm that the composition of hPL is affected by pathogen reduction treatment, but cell culture performance remains unaffected (Viau et al., 2017; González et al., 2022). There is some evidence of failure especially regarding non-enveloped viruses and the application of complementary reduction methods is recommended for large pooling strategies (Blümel et al., 2020). Therefore, careful selection and application of pathogen reduction treatments can ensure the safety and effectiveness of repurposing large volumes of expired PCs and enable further studies to better compare hPL and FBS in HCC.