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Cell therapeutics are emerging as a viable modality to treat challenging diseases such as cancer, organ degeneration, or (auto)immune diseases resulting in an increased demand for their large-scale, high-quality production. Currently, fetal bovine serum (FBS) is the most widely used growth factor supplement for the expansion of human cell therapy products. However, there has been a strong impetus from regulatory agencies and biomedical professionals in the field to develop methods for cell expansion that do not utilize animal products in their production process.1 Human platelet lysate (hPL) has been identified as a possible growth supplement contender—rich in growth factors and produced in most cases from expired platelets (PLTs)—that can be used to replace FBS. What is hPL? hPL is essentially the product of lysing human PLTs typically involving freezing and thawing.2 There are several protocols that have been generated for hPL production, some of which are proprietary. The general approach is to generate large pools of hPL to limit batch-to-batch variation because PLTs are by nature one of the most heterogeneous components of blood. hPL is routinely produced from PLT-rich plasma (PRP) or expired PLTs collected via apheresis.3, 4 Multiple freeze/thaw cycles cause the PLT membranes to burst and release trophogens and growth factors resulting in a lysate. Aggregates, debris, and PLT fragments are removed by centrifugation.3, 4 Human PLT lysate can be derived from autologous (same donor) collections, which may minimize the risk of immunologic reactions or infections; however, most efforts have focused on preparing allogeneic pooled batches from blood donations from a large number (50-100) of different individuals. The use of hPL in standard cell culture protocols could prove to be a promising tool for further development of cell therapy products in animal protein–free systems. There are a number of challenges and unknown factors to investigate in the development of hPL as a growth factor supplement starting with its heterogeneous composition. A variety of factors can affect the composition and bioactivity of hPL including the plasma content, growth factor content, age and gender of the donor, PLT counts and/or volume, production process, heparin and/or anticoagulants, mRNAs, metabolites, blood group, storage conditions of the unit, extracellular vesicle content, and potential treatment to reduce and/or inactivate pathogens.5-8 AABB and International Society of Cell Therapy (ISCT) established a joint working group on hPL to identify gaps of knowledge that could eventually lead to developing and defining recommendations for standardized manufacturing and quality control (QC) of hPL. This review is the first result of this working group, summarizing identified gaps of knowledge to pave the way for necessary consensus. Fetal bovine serum has been used for decades as a supplement in a wide range of cell culture applications in cell-based research, drug discovery, diagnostics, toxicity testing, cell therapy, in vitro fertilization, human and animal vaccine production, and biopharmaceutical manufacturing.9 FBS has been considered rather unique in its universal capacity to support the growth of multiple cell types in culture. Although widely used, there are various reasons to find suitable alternatives to the use of FBS. Fetal bovine serum adds proteins, attachment factors, growth factors, enzymes, protease inhibitors, hormones, vitamins, trace elements, and numerous others to media to help cells adhere, survive, and proliferate; however, the relevant factors contributing to promotion of cell growth still remain largely unknown.9 Replacing ill-defined serum with chemically defined medium appears beneficial, yet these media can be complicated to develop10 and specific formulation adjustments may be necessary to support the growth of different cell types. Serum-free, animal component–free, and or chemically defined media have been developed.11 There are databases (e.g., https://fcs-free.org/fcs-database or http://www.sefrec.com/) that list different cell types, products, and resources; however, to date only a small number of protocols are available where these can be applied. Despite international standards set by the International Serum Industry Association,12 there is strong concern regarding the inhumane method of procuring FBS by cardiac puncture without anesthesia.13, 14 There is an increasing market for FBS that is dependent in large part on the beef processing industry.15, 16 Increasing demand from life science and pharmaceutical users, restricted supply due to climate changes, and reduced cattle inventories have impacted costs and have led to questionable practices in production. Cases where bovine serum albumin, water, and/or cell growth–promoting additives have been used to blend the FBS have been reported.15 Xenoantigens can serve as immunogens that compromise therapeutic efficacy.16-19 Sundin and colleagues described the induction of alloantibodies against FBS proteins in three of 12 individuals who received matched or mismatched mesenchymal stromal cells (MSCs). Similar patterns of FBS reactivity were observed in healthy subjects, indicating a degree of preimmunization against FBS, which could compromise the therapeutic efficacy of cell products.20 The potential presence of extraneous agents in FBS is considered a risk to the safety of biologic medicinal products, although to the best of our knowledge no cases have been reported. Regulatory authorities clarified that “the use of adequately controlled bovine sera is allowed.” Reinhardt and colleagues17 specify that FBS must be controlled according to the European Pharmacopoeia, 7th ed, Monograph 01/2008:2262 Bovine Serum,18 and manufacturing shall involve virus testing and inactivation (e.g., irradiation at a dose higher than 30 kGy). The risk of transmitting animal spongiform encephalopathy agents, in particular, has prompted the search for alternative supplements from nonanimal origin.16-18 Regulatory authorities released guidelines or notes of guidance for the manufacture of human medicinal products. Initially the European Medicines Agency recommended, “When a manufacturer has a choice between the use of ruminant or non-ruminant material, the use of non-ruminant material is preferred.” If there are no alternatives to FBS available, specific risk assessment is needed to account for extraneous agents and lot-to-lot reproducibility.19, 21 Human PLT lysate was proposed as a xeno-free culture supplement in the 1980s to expand rabbit articular chondrocytes and cell lines from a variety of tissues and tumors.22-24 In 2003, Lucarelli and coworkers,25 while investigating the effect of PRP released by PLT gel on stromal stem cell proliferation and differentiation, observed significant growth-promoting capabilities of hPL. Doucet and coworkers1 introduced hPL as a safe substitute for FBS in MSC culture, followed by intense research in the field of MSC culture and documented by numerous publications.5, 26 Muraglia and coworkers31 reported on various preparations of hPL for MSC and human articular cartilage, namely “(i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s).” Not too surprisingly, the hPL derived from plasma (as it lacks PLT factors) failed to support growth of both MSC and chondrocytes, but supported that of certain tumor cell lines, whereas the v-PL was fully active.27 Human PLT lysate has been tested on a variety of cell types as exemplified in Table 1. Here we will focus on MSCs. MSCs have stromal activity; that is, they contribute to the hematopoietic stem cell niche, they are potent immunomodulators interacting with different immune cells, and they produce and secrete various trophic factors and extracellular vesicles that likely mediate their regenerative potential.42, 43 hPL can safely replace FBS for clinical-scale MSC manufacturing: Data show that hPL can support MSC proliferation, maintain colony-forming units–fibroblasts content during in vitro culture and hold chromosomal stability comparable to or in some cases better than FBS (reviewed in Astori et al.,5 Bieback,6 and Tan et al.26). MSCs cultivated in hPL show enhanced osteoblastic differentiation potential44, 45 and reduced adipogenic differentiation capacity45 indicating that using hPL may be a superior option in MSCs cultured for bone regeneration. However, reduced, similar, or increased immunomodulatory MSC activities have been reported comparing hPL and FBS-cultivated MSCs.46-48 This functional variability emphasizes the need for studies comparing FBS and hPL (different hPL products) in MSC (or other cell types of interest) culture. Endothelial cells Endothelial progenitor cells/endothelial colony-forming cells Promotion of cell growth Compared to FBS: maintained phenotype, functional characteristics like tube-like vessel formation on matrigel and metabolic state Renisch et al.,34 Siegel et al.,35 Tasev et al.36 Hofbauer et al.37 Numerous clinical trials are currently with MSCs for a variety of MSCs and in FBS were tested in using MSCs and cultivated in hPL have been In the autologous MSCs to be safe as no were and that MSC is and safe in with no or against of MSCs from a donor, of the and the hematopoietic stem cell donor) were and clinical were observed in of A different of with MSCs in medium cells a reduced immunomodulatory In vitro regarding effect on and proliferation support this is unknown the cell culture supplement cell in the of clinical a manufacturing MSCs for the treatment of reported that of the on hPL used hPL produced in a blood and used available to of that still use Human PLT lysate preparations in these studies likely on the way they were that the presence of in hPL may the capacity of and others have observed comparing and MSCs using FBS, human and different PLT preparations lysate or In our MSCs from only in the first using hPL. MSC proliferation was reduced to FBS. Human serum or PLT in supported expansion of MSC from the by FBS. MSCs derived from however, in hPL and reduced proliferation in human serum and that MSCs from different different of their cell culture identified a factors that in comparing hPL and using a reduced during the in a growth-promoting effect on MSCs from and studies may help to identify the growth factors for specific cells and these factors can be to the medium A better of the could production of a better defined Human PLT lysate can be used in an autologous or allogeneic we focus on allogeneic hPL. The number of cells typically needed to clinical than the typically from one blood or PLT and it is challenging to multiple blood donations or from a or is hPL is to has lot-to-lot can be and is than autologous hPL. has been the supplement of choice in most pools have been that in be however, may be by a number of A pooled hPL products have been using than but the of pools is an large have been by the regulatory of of the Regulatory have the to a of 16 and serum and/or testing according to guidelines on blood quality and The European in that of the risk of transmitting agents from pooled pooled or other derived from pooled human blood or is to limit the number of donations which are methods for of are during production, where This prompted to the and or potential quality of on of for PLT for hPL production must not be than for blood product virus virus virus and although can be tested for on and hPL produced from different have growth factor content on cell The age of appears to affect quality of hPL and its effect on cell and that the proliferation of MSCs was higher with hPL produced from than with hPL produced from which was not with growth factor hPL from increased the of the no studies have potential of hPL to or which may hPL quality be needed to to hPL batch-to-batch There may be of blood group on hPL is that hPL from blood group PLTs in plasma may be the product of as potential with and on cells and in the are However, it has been in several studies that hPL derived from blood group PLTs cell proliferation without cell (reviewed in et al.26). Tan and the of PLT cell in hPL by for the cell and the This that blood group PLTs could be used as a starting material for hPL production without safety involving on PLT cell The for PLT including the of the the way they are and the conditions which they are can the quality of the hPL can be generated from or PLT in et The of blood cells in on the PLTs are which can affect the content in PLTs are can be in plasma or in a of plasma and PLT storage and which can cause and that growth factor content is higher in PRP from with that derived from However, and not find significant in growth factor content or on MSC proliferation between hPL produced from derived from or from this of studies comparing the of different hPL products on various cell types using standardized (e.g., promotion of cell proliferation and/or growth factor are and expired PLTs can be used as the starting material for hPL however, there is variability on the of the on can be for to 4 to The use of during or their life will on the or where the hPL will be of this potential for it be to the for hPL PLTs used for are at however, it has been that growth factors are not in plasma at that PLTs could be a better quality product as a starting material for hPL. The collected do not that hPL from expired has hPL from and that hPL from PLTs was as for proliferation as hPL from PLTs and as as growth factor and growth factor and hPL from and no significant in their growth-promoting on MSCs. and these and further that hPL from PLTs was not different to hPL produced from PLTs in their effect on MSC proliferation, differentiation and studies support the use of PLTs and potential of donations for that can serve as alternative potent and safe media supplements for cell is that to of produced in this for hPL manufacture be an and to support cell manufacturing without at and blood be to conditions to best quality PLT Cell therapy is a field and the of hPL that will be needed for cell-based products in the will be it is to have between used for hPL production. The use of expired or of of the of and blood be the for manufacturing hPL in to for cell Human PLT lysate can be generated from by and PLT with or review on the different production methods for hPL has been In to the use of different of variability is in the various protocols used to manufacture hPL reported in the the to best The protocols used for hPL production could affect growth factor and the of the we that PLT by and hPL by cycles different on MSC with the of standardized hPL has for in vitro culture of different cell 26 Although methods have been tested (reviewed in et the most for PLT is from one to freeze/thaw This is a and approach to produce however, the number of freeze/thaw cycles to be freeze/thaw cycles can result in growth factor while an number of freeze/thaw cycles could growth factor due to PLT and one and freezing cycles to PLTs and no significant on the of hPL on MSC and and that the cell was cycles were that one to cycles be to release growth factors without their The reported used for freeze/thaw cycles from for freezing and for and different that the growth factor were freezing at and at was can be in the in which they are (e.g., PLT or a of or can be and in or The of plasma in the product appears to be as plasma growth factors that can supplement from one has that hPL with plasma is than hPL in cell The of heparin to cell culture medium hPL is to however, heparin is derived from is a xeno-free and some cases of to heparin have been of cell proliferation at heparin and possible with and of bone cells to has been have been introduced in the production of hPL. The use of hPL be where no heparin is the presence of that is in hPL heparin is Although hPL be or can the of growth factors and factors and proteins such as and that whereas hPL was for MSC proliferation, hPL in higher MSC There were no in the of growth factors such as growth growth factor and growth factor between and on in and were observed in both and or not this can serve as a of quality to be of these be for to different cell types. heparin have been which could replace heparin for a hPL supplement without the need to hPL is which or and for can hPL be to these and that hPL could be at for at while a of it has been that hPL are at for and that hPL could be maintained for 4 at without significant of The of hPL in cell culture media between studies and cell types, from to with to the most This will on the starting PLT which between to in of hPL production is currently a factor content due to the use of protocols and additives such as plasma for hPL and studies in which different hPL products by are used in to cell growth of different cell types, and where of the cells are are needed to on the field and the from FBS to hPL in clinical and of the studies have been on but other cell types of potential for cell therapy and research, such as stem cells, and stem cells, be tested FBS with hPL of hPL standardized protocols according to manufacturing guidelines and There is a need to an international on quality for hPL products for cell The on human platelet lysate for cell the variability in production and quality 4 The European general on of for the production of cell-based and therapy medicinal products for human some in this that only adequately tested for agents according to and/or be A risk assessment for safety and spongiform be according to European and to quality of the and biologic be must the of the material and it from other products. that can be used for hPL are content, and of specific growth factor in hPL have been it is still not which growth factors are for cell growth of a specific cell of the of factors to PLT can be a be by and can be tested by the cell growth–promoting effect of the specific on the cell of The can be to manufacturing and potential of the release be different on the PLT as quality standards for PLT donations are different between and in such as testing have blood of diseases like or International and climate may such as or virus blood with a with of such may challenges in manufacturing safe blood products, the testing are not the most method has its with to yet or or of during can cause of blood these for have been that can the potential and content of blood has been by the European for pooled for the production of cell-based or therapy medicinal date the for PLT and and most The using their with and whereas with with and that have been to reduce the of and and in and must be however, there is no need to or its In treatment it is only for the inactivation of treatment to the release of and from hPL can the immune to an in a that the release of immunomodulatory factors from is to too do not only the of but affect PLTs and their treatment has been to the PLT A comparing on that but not PLT and and that this was with PLT of on the PLT are for proteins to the of vesicle or PLT A review of studies reported on PLT proteins by proteins by and proteins by The of and to and/or and proteins are similar, while the of is higher for and/or to This to or the potential effect of may have a effect on of irradiation on hPL in inactivation of bovine and treatment has been to have some on growth factor content in hPL such as of however, factors to date remain or are to be increased as for the presence of treatment treatment of the proliferation of MSC cultured in medium with hPL that was from these MSCs cultured with or their superior proliferation to MSC with of and assessment of the in vitro differentiation potential are part of the MSC The use of hPL produced from or not the of the In these studies the tested were possible on other on the MSC be at this of on MSC in vitro are studies not an of or on MSC or differentiation in vitro and MSC potential to the proliferation of immune cells in as as on their formation and In one that MSC from reported of on of in and in MSCs in vitro In the MSCs cultured with immune cell proliferation in factors, but the irradiation not the content of various growth factors for in hPL or its for MSC culture and In hPL production, is on PLT What could be on the hPL product can affect the of is to that the of PLT at in to of as the and/or the are not interacting with cells or PLTs but with their released proteins and studies are needed to better on to identify the for the hPL and to at the of the hPL manufacturing Human PLT lysate has been as a suitable alternative to FBS (e.g., in clinical-scale manufacturing of hPL production and efforts to it as a growth supplement for cell therapy products multiple challenges are likely to A yet to be clarified is is the for production of hPL? The product from to and has its may that a hPL product is for expansion of one cell and one clinical but not the The of hPL product is and dependent on of production. The of the hPL product may need to be cell product and dependent and may to cells are is of to the of expansion of the cell hPL studies have been on cells that are in cell or culture The field of cell therapy production is using for expansion to costs and has yet to be hPL can be used in some of these in use but is an option of further of are better defining cell therapy products. The hPL product may contribute to and characteristics of the cell product which can the therapeutic of the cells, and The and of the cells produced with a specific hPL including hPL products, is a and an for further is that is for the production of a safe product for clinical hPL have an capacity to support cell expansion and maintain cell There are challenges with of starting material for hPL the the demand for hPL? it be widely as a number of cell products to market and the demand hPL be adequately not only for cell-based but for (e.g., vaccine production or hPL for blood who are PLTs for PLT the use of PLTs for hPL manufacture is to PLTs with their hPL to be and produced and this will product product and will be to There are that human or PLT lysate may be an alternative to however, there are a number of regarding hPL manufacturing and manufacturing of quality with to and not safety are the to be of cell growth promotion components that are for the cell of the relevant composition can be a or chemically defined medium The best hPL product for a specific cell the quality and composition of hPL the of the cell product and it is clinical dependent hPL factors expansion of cell products manufacturing hPL quality quality for hPL release there is an of starting material for hPL to the of the cell therapy as increasing of products are by regulatory agencies affect hPL quality a is The of the The manufacturing protocols to identify quality and they are in however, there is not available, which for recommendations for standardized manufacturing and This the first of the and the Human gaps of knowledge and to these to develop for and of hPL as cell culture This has been on of the and Human and the of of the joint working AABB for for of the and by is of a for the use different media on The have no other or in the products or described in this