The body tissue aging is linked to a progressive loss of homeostasis and regenerative potential of the organism. Some studies showed a “rejuvenating” effect of exposing aging tissues to a young environment.

In the early 1970s, scientists began to join rodents of different ages (heterochronic parabiosis) and discovered that, in response to the young organisms, the old animals lived longer compared to isochrony old age-matched conjoint animals. It was hypothesized that systemic alterations in the global regulators of tissue physiology present in the blood may coordinate this process. Conboy et al. showed that the rejuvenating effect of heterochronic parabiosis on skeletal muscle could be recapitulated in vitro by exposing aging satellite cells of mice to young serum (Conboy et al., 2005). On the contrary, the deleterious effect of an aged environment on stem cell functionality was observed in parabiotic pairings (Villeda et al., 2011). The same Stanford University group, by the injection of young plasma in mice, obtained an effect comparable to heterochronic parabiosis in reversing age-related weakening of cognitive function in spatial–memory tests (Villeda et al., 2014). The blood of young mice appeared to rejuvenate all organs of the old mice, making the animals healthier and cognitively fit. A more recent report showed a beneficial effect of young animal plasmas in vivo in a mouse model of Alzheimer's disease (Zhao et al., 2020). Finally, human umbilical cord blood plasma was shown to revitalize hippocampal function in aged mice (Castellano et al., 2017) and to promote neurogenesis while reducing inflammation in a rat model of ischemic stroke (Yoo et al., 2016).

The discovery of tissue rejuvenation as a consequence of the transfusion of blood from young to aged animals raised incredible hopes of slowing down the senescence process in humans, as well as to prevent or cure age-related diseases such as cancer, brittle bones, and Alzheimer’s. However, obtaining the same results in humans is far from straightforward, and uncertainties still exist in a major part of the scientific and medical community. A recent publication reported the results of a randomized clinical trial performed to assess the safety, tolerability, and feasibility of four weekly infusions of young, fresh, frozen plasma (donors aged 18–30 years) to treat Alzheimer’s disease (AD) symptoms in 18 individuals aged 50–90 years (mean age 74 years) (Sha et al., 2019). Although no amelioration of neurological symptoms could be determined, the study showed no adverse events, indicating that plasma therapy was safe and well tolerated at least under a short-term protocol. Although most results were not significant, two metrics–the Functional Activities Questionnaire and the Alzheimer’s Disease Cooperative Study–Activities of Daily Living Inventory–showed a significant improvement, following plasma treatment. However, the study design had some shortcomings. Half of the AD patients were not blinded and knew which treatment they were receiving, and the significance reported for these measures reflected a within-group comparison (before vs. after treatment), as opposed to plasma vs. placebo. A handful of additional clinical trials, such as trials on the potential benefits of young plasma for AD and Parkinson’s disease (NCT03765762 and NCT03713957) and a study on the use of umbilical cord blood for treating age-related frailty (NCT02418013), are currently being performed or were recently completed. However, these studies are small and of limited duration. Often, they lack placebo controls, underlining their preliminary nature. Overall, to date, there is no real convincing evidence to support the effectiveness of young plasma as an anti-aging treatment in humans.

Unfortunately, the possibility of tissue rejuvenation by young plasma injection also raised the interest of some for-profit stem cell clinics and biotech companies, which started to transfuse plasma to humans according to scientifically dubious protocols regarding the amount, number, and timing of infusions. In February 2019, the FDA issued a general warning to consumers: plasma infusion from young people has not yet provided enough proven clinical benefit against aging and/or its diseases to justify its adoption on many individuals. One of these companies claimed that they did not need approval by the FDA because plasma transfusion is a well-established clinical practice and continued the treatments.

The results obtained to date by the injection of young plasma in aged rodents and humans, while confirming the great interest in the potential rejuvenation capability of this plasma, on the other hand underline the need for additional investigations before one can seriously think of a large-scale treatment of human subjects. Required investigations include the development of assays to measure the biological activity of the different human plasmas and to select the more efficacious plasmas.

We reported that in primary cultures of cells obtained from tissue biopsies, the platelet lysate activated signal transduction pathways distinctive of proliferation and was required for the re-entry of cells in the cell cycle, while the non-cellular component of the blood supported the cell division. On the contrary, in constitutively activated cell lines, plasma or plasma-derived serum sustained cell proliferation, regardless of the co-presence of the platelet lysate (Muraglia et al., 2017). Therefore, to be able to measure the capacity of plasma to induce cell proliferation appears highly relevant to assess the clinical potential of individual plasmas. We started from these observations to develop and propose a simple method to measure and compare the capability of different plasmas in supporting cell proliferation, thus determining their potential use or nonuse for clinical applications. The adoption of standardized cell lines and culture conditions and the identification of absolute indicator values should allow the direct comparison of unlimited plasmas also when tested at different times. The article is focused on the potential use of young plasma as a “rejuvenating” agent. However, the selection of more efficacious plasmas could also be important for the preparation of allogenic Platelet-Rich Plasma (PRP) to be used for the healing of different tissue damages, a therapeutic approach which has proven to be beneficial in several clinical trials.

Plasma was derived from blood samples of young (age: 18–25 years; average age: 21.6 ± 1.8 years; 8 males and 12 females) and old subjects (age: 75–99 years; average age: 85.6 ± 6.1 years; 10 males and 9 females) obtained from the Transfusion Center and the Emergency and Geriatric Clinic of “Ospedale Policlinico” San Martino, Genova, Italy. A total of 39 plasmas were tested since one plasma was tested in both types of cell cultures. More specifically, plasmas from young subjects were collected from regular blood donors. All young blood donors were in good health, according to the criteria followed by the Transfusion Center, to allow blood donation. Blood donation is not allowed after 60 years of age. Therefore, the plasmas of the old subjects utilized were collected from voluntary donors and hospitalized patients. Plasmas to support the proliferation of MDA cells were obtained from the following subjects: three subjects from the emergency department because of a fall associated in one case to head trauma/presence of deep thrombosis/hypertension and subarachnoid hemorrhage; three subjects temporarily hospitalized in the geriatric clinic because of a mental illness associated with psoriasis with allergic asthma, diabetes, and acute heart failure, respectively; and four subjects hospitalized for a femur fracture. No significant differences were observed between plasmas from fallen subjects and the patients with mental illness. The old plasmas utilized for the HFFF2 cultures were from healthy voluntary donors (n = 2), fallen/fractured individuals (n = 6), and a patient with osteoporosis associated with intestinal perforation (n = 1). All patients were of Caucasian origin.

No specific clustering was observed in cell duplication values obtained from the patients of the different subgroups. All participants provided written informed consent for their participation in the study. Whole blood was harvested in K₃-EDTA-containing sampling tubes and was centrifuged at 1,600 g for 15 min at 4°C. The supernatant, corresponding to the plasma fraction, was recovered, divided into aliquots and stored at −80°C until further use.

Human serum from the whole blood, i.e., a combination of the platelet content and the non-cellular component of blood, was adopted as the cell culture medium supplement to replace fetal calf serum (FCS), with even higher efficiency. In constitutively activated cell lines, plasma or plasma-derived serum sustained cell proliferation, regardless of the co-presence of the platelet lysate (Muraglia et al., 2017). The human breast cancer-derived MDA cells maintained in culture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS were plated at a concentration of 1 × 10³ cells per well in 24-well plates. After three days of culture, cells were gently rinsed with phosphate-buffered saline (PBS), starved for 2 h in serum-free medium and then treated with fresh medium containing the tested plasma at the concentration of 0.1, 0.5, 1 and 2.5%. To avoid the activation of plasma clotting by cell-released proteases within the Petri dishes, 5 UI/mL sodium heparin was added to the medium. From the day of the medium change up to day 5 of the culture, the cells were detached daily by trypsin–EDTA digestion from triplicate wells of each plasma concentration series and counted.

Human Foetal Foreskin Fibroblast (HFFF2) cells suspended in DMEM containing 10% FCS were plated at a concentration of 2 × 10³ in wells of 24-well plates. The cells were plated in triplicate wells for each of the two chosen plasma concentrations (2.5% and 0.1%) and the two times to be tested (days 0 and 6). After 48 h from plating, the medium was removed, the cells were rinsed with PBS, then transferred to a serum-free medium for 1 h, and finally cultured with a fresh medium containing the plasma. Given the lower proliferation rate of the HFFF2 cell line than that of MDA cells, we stopped the cultures, and we determined the number of cell doublings 6 days from the change of medium instead of 5 days. MDA and HFFF2 cultures were performed at 37°C and 5% CO₂.

To detect possible cytokine and inflammatory protein concentration differences between young and old plasmas, we used the Human XL Cytokine Array Kit (Catalog Number: ARY022B; R&D Systems, Inc., Minneapolis, United States), a tool used for the parallel determination of the relative levels of 105 selected human proteins. On each array, we tested plasma from both a young and an old individual. Loaded volumes of old and young plasmas contained 200 μg of proteins. The protein concentration was assessed by the Pierce™ BCA Protein Assay (Thermo Scientific). The protein signal was obtained by a UVITEC chemiluminescence imaging system and counted by ImageJ using the appropriate macro (Klemm, 2020).

Differences between cell culture conditions were assessed by the non-parametric Mann–Whitney test. The protein signal differences were calculated by Student’s t-test. Statistical significance was accepted for any p-value ≤ 0.05. Analyses were performed using GraphPad Prism 9.4.0 software. Cytokine array data were analyzed using a custom R script including Bioconductor packages. In particular, log-transformed data were normalized by quantile normalization using the preprocessCore package. Then, differential enrichment analysis was performed using limma package (Ritchie et al., 2015). p-values derived from Bayesian statistics were adjusted by the Benjamini and Hochberg method.

On the assumption that the young blood effect may be attributed to specific proteins and growth factors more abundant in plasmas of younger individuals or the presence of inhibitors in the old plasmas, to detect possible cytokine and inflammatory protein concentration differences between young and old plasmas, we utilized four arrays from two different batches to analyze eight plasmas (four young and four old). Data were first analyzed by a volcano plot, a type of scatter plot that shows statistical significance (p-value) versus folds of change. All the data had a quite noisy distribution, so we used various pre-treatment approaches. We ended up by separately considering data from the two array batches, removing batch effects and then performing quantile normalization. The obtained volcano plot is shown in Figure 3. On the left side of the graph, one can see young plasma-enriched proteins, while on the right side are those enriched in the old plasmas. Statistically significantly expressed proteins are shown. Five proteins emerged as increased in the old plasmas: the C-reactive protein (CRP); an acute-phase protein produced by the liver; the circulating concentration of which increases in response to inflammation; the IGFBP2, a factor binding to insulin-like growth factors, thus allowing their free movement to target tissues; chitinase-3-like 1, a non-enzymatic protein playing a major role after a tissue injury; and two proteins synthesized by the cells in response to inflammation, cystatin C and lipocalin 2. Proteins present at a higher concentration in the young plasmas and decreased in the old were the vitamin D-binding protein, a multifunctional protein, that also binds to fatty acids and actin monomers, preventing their polymerization that could be detrimental to the circulatory system; and apolipoprotein A-1, an anti-inflammatory component of HDL, involved in cholesterol efflux.

As an alternative method of analysis, for each couple of plasmas (young and old) tested on the same array, we determined the fold increase or decrease in the protein concentration in the old plasma compared to the young plasma. Then, the results from the four different arrays were combined and the statistical significance evaluated. A summary is shown in Figure 4. The proteins with the highest concentration increases observed in the old plasmas were again CRP (approximately 30-fold increase, p = 0.03), IGFBP2 (approximately 20-fold increase, p = 0.01), and chitinase-3-like 1 (more than 6-fold increase, p = 0.02) (Figure 4A). More proteins significantly increased (between 2 and 4-fold, p = less than 0.04) in old plasmas, in addition to the already identified cystatin-C and lipocalin 2, including several cytokines involved in inflammation such as tumor necrosis factor alpha (TNF-α), with pleiotropic effects on various cell types, GDF-15, first identified as macrophage inhibitory cytokine-1, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1ra, IL-3, IL-18Bpa, also known as interferon-inducing factor, IL-27, interferon-inducible T-cell alpha chemoattractant (I-TAC), and macrophage inflammatory protein-1 alpha (MIP-1alpha/CCL3). Other proteins that increased in old plasmas and were synthesized in response to inflammation were osteopontin, resistin, and vascular cell adhesion molecule 1 (VCAM-1) (Figure 4B). Considering the increase in the old plasmas of these proteins related to inflammation, one could hypothesize a role for them as inhibitors of the plasma cell proliferation stimulatory activity. However, the coincidence of the increase in the inflammatory cytokines and proteins and the decrease in the cell proliferation induction does not necessarily imply a cause–effect relationship. Note the extreme variability in the concentration of these proteins in the plasmas of old subjects compared to young subjects, suggesting major concentration variations in the plasmas from different old individuals. Possibly, because of a bias in their selection, among the 105 proteins present on the array, none showed a significant decrease of at least 2-fold in the old plasmas. The only proteins with a significant decrease revealed in the t-test were the vitamin D-binding protein and the apolipoprotein A-1 (Figure 4C). However, the observed decreases were limited to 42% and 13%, respectively.