Guiotto et al. J Transl Med (2020) 18:351 https://doi.org/10.1186/s12967-020-02489-4 Journal of Translational Medicine

REVIEW Open Access Human lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review M. Guiotto1,2* , W. Rafoul1, A. M. Hart2,3, M. O. Riehle2† and P. G. di Summa1†

Abstract Background: Foetal bovine serum (FBS), is the most commonly used culture medium additive for in vitro cultures, despite its undefned composition, its potential immunogenicity and possible prion/zoonotic transmission. For these reasons, signifcant eforts have been targeted at fnding a substitute, such as serum free-media or human platelet- lysates (hPL). Our aim is to critically appraise the state-of-art for hPL in the published literature, comparing its impact with FBS. Materials and methods: In June 2019 a systematic search of the entire Web of Science, Medline and PubMed database was performed with the following search terms: (mesenchymal stem cells) AND (fetal bovine serum OR fetal bovine calf) AND (human platelet lysate). Excluded from this search were review articles that were published before 2005, manuscripts in which mesenchymal stem cells (MSCs) were not from human sources, and when the FBS controls were missing. Results: Based on our search algorithm, 56 papers were selected. A review of these papers indicated that hMSCs cul- tured with hPL showed a spindle-shaped elongated morphology, had higher proliferation indexes, similar cluster of diferentiation (CD) markers and no signifcant variation in diferentiation lineage (osteocyte, adipocyte, and chondro- cyte) compared to those cultured with FBS. Main sources of primary hMSCs were either fat tissue or bone marrow; in a few studies cells isolated from alternative sources showed no relevant diference in their response. Conclusion: Despite the diference in medium choice and a lack of standardization of hPL manufacturing, the major- ity of publications support that hPL was at least as efective as FBS in promoting adhesion, survival and proliferation of hMSCs. We conclude that hPL should be considered a viable alternative to FBS in hMSCs culture—especially with a view for their clinical use. Keywords: Human platelet lysate, Foetal bovine serum, Mesenchymal stem cells, Adipose derived stem cells, Cell therapy

Te word “mesenchymal stem cells” or MSCs and “stro- mal cells” are here used as synonym despite the recent *Correspondence: [email protected]; [email protected] distinction between the tissue-specifc (e.g. adipose, †M. O. Riehle and P. G. di Summa contributed equally and share the co- bone, cartilage, …) stem/progenitor cells and the old last authorship 2 Centre for the Cellular Microenvironment, University of Glasgow, generic term mesenchymal stem cells [1]. Glasgow, UK Full list of author information is available at the end of the article

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Background In addition, FBS enables expansion of human cell cul- Te application of cellular therapies is growing enor- tures in vitro and supports trilineage diferentiation mously in a wide range of medical felds, often using potential (osteogenic, adipogenic and chondrogenic) of human mesenchymal stromal/stem cells (hMSCs). hMSCs. Terefore, it has, until now, been the media sup- In the clinic hMSCs are currently used for bone mar- plement of choice in a wide range of proto- row transplantation, to treat bone or cartilage defects, cols [6]. myocardial infarction and to manage graft versus host Despite that, the composition of FBS medium addi- disease. Potentially for regenerative purposes, hMSCs tive is not completely determined, with a wide hetero- could also be applied in combination with tissue engi- geneity among samples [6]. Furthermore, when used in neering strategies to treat musculoskeletal and neurologi- a translational setting, anaphylactoid reactions and the cal disorders [2]. risk of zoonoses transmission were reported [7, 8]. Tese hMSCs are non-hematopoietic progenitors, with an reports suggest that using FBS is, in essence, not compli- ability to diferentiate along mesenchymal and non-mes- ant with the principles of GMP, because it may afect the enchymal lines, such as adipose, chondrocyte or osteo- safety and efcacy of cell therapy [9–11]. blast lineages. Bone marrow, umbilical cord blood, dental For these reasons, signifcant eforts are targeted at pulp and adipose tissue are all potential sources for autol- fnding a substitute for animal serum such as serum free ogous hMSCs in stem cell-based therapies. However, media or platelet derivates [12]. Alternatives to FBS and bone marrow (BM-hMSCs) and adipose derived mesen- serum free media, that could help for the translation of chymal stromal/stem cells (ADSCs) are more popular. cell therapy to the clinic, are human serum (hS), plate- Isolation of BM-hMSCs is a complex and painful pro- let-rich plasma (PRP) and human platelet lysate (hPL). cess for the patient, with a low cell yield, while ADSCs When allogeneic hS is implemented in cell expansion extraction by liposuction is a less invasive procedure with hMSCs proliferation rate is reduced and the time it takes minimal patient discomfort. Moreover, adipose tissue for cells to reach confuence extended compared to the has a 500 times higher number of stem cells than bone standard FBS cultures [13]. In addition, the concentra- marrow per unit of tissue (weight/volume), therefore it tion of growth factors seems to be limited in both hS and has recently been recognized as the source of choice for FBS, and some authors found that the debris, that forms hMSCs [3]. during PRP production, could slow down cell expansion Despite that, the clinical applications of hMSCs is hin- [6, 14]. However, the relative advantages of these difer- dered by their availability, because the number of cells ent culture media additives are still widely debated and that can be safely isolated per unit tissue volume is lim- recent studies showed that particularly hPL could be a ited. Tus, hMSCs need to be expanded and may have to valid substitute for FBS, retaining stem cell phenotype be diferentiated ex vivo before their therapeutic applica- (positive expression of CD (cluster of diferentiation) tion, requiring in vitro culture. Te entire process (isola- CD73, CD90, CD105, but negative for CD34 and CD45) tion, in vitro culture and eventual diferentiation) must and multilineage diferentiation capacity (osteogenic, adi- respect the principles of good manufacturing practice pogenic and chondrogenic) of hMSCs [15–17]. (GMP) in order to minimize risks and maximize the ben- hPL can be easily prepared using at least three difer- efts of cell therapy [4, 5]. ent manufacturing methods, such as , “bufy coat” (BC) or platelet-rich plasma (PRP). Releasing Foetal bovine serum and human platelet lysate the growth factors from the represents the key Foetal bovine serum (FBS), also called foetal calf serum, point in hPL production: again, four diferent methods represents the most common serum additive for in vitro are available to attain platelet lysis: repeated freeze/thaw usage, which supports adhesion, growth and prolifera- (FT) cycles, direct platelet activation, sonifcation alone tion of a wide spectrum of diferent cells. FBS usually is or in combination with thermolysis and solvent/deter- extracted from foetal bovine blood, being collected after gent (S/D) treatment. slaughter of pregnant cows under sterile conditions. After How these means of preparation infuence the bio- clotting, centrifugation steps are carried out to separate chemical and functional properties of hPL, has not yet its cellular components, before fltration steps (normally been sufciently investigated. hPL has the higher concen- 100 nm pore size) are applied to remove potential bacte- tration of growth factors (GFs), such as platelet-derived rial and viral contaminations. FBS is well known for its growth factors (PDGF-AB), basic fbroblast growth factor cost efectiveness and its richness in adhesion molecules, (bFGF), transforming growth factor beta (TGF-ß1), insu- growth factors, micronutrients and hormones which pro- lin-like growth factor 1 (IGF-1) and vascular endothelial mote attachment, growth and proliferation of mamma- growth factor (VEGF), than any other cell culture sup- lian cells. plements including PRP and FBS: this may be the reason Guiotto et al. J Transl Med (2020) 18:351 Page 3 of 14

why the majority of recent reports agree that hPL sup- (1000g for 10 min at 21–22 °C) to separate the blood cells ports cell expansion to a higher degree, compared to FBS, from the upper layer consisting of platelets mixed with hS and PRP [11, 13, 18–20]. Being a human blood deri- plasma. In plateletpheresis, blood, taken directly from a vate, allogeneic hPL requires to be tested for HIV, hepa- donor, and processed by an machine that uses titis B, C and other potential viral infections [6, 13, 21]. centrifugation to remove a selected component from the From a physiological perspective, platelets contain a blood and returns the remainder to the donor. great variety of growth factors, cytokines, proteins and Te resulting platelet concentrates are stored for factors that support clotting. When platelets are lysed, 5–7 days at 22 °C under agitation [29]. To avoid bacterial they release their content consisting of albumin, folate, contamination, platelet units should be stored at 22 °C for vitamin B12, glucose, triglycerides and cholesterol (less up to a maximum of 7 days from the time of donation. If concentrated in comparison to FBS), immunoglobulins they are not used with a clinical purpose, they can be fro- (mainly IgG, in higher concentration compared to FBS) zen and subsequently be used for hPL production. How- and other proteins that also contribute to balance media ever, no data exist that clearly state the maximum period colloid pressure. In addition, platelets contain numerous of time, beyond 7 days, for which platelet units can still growth factors, mainly TGF-ß, platelet-derived growth be used to obtain an efcient and safe hPL product [30]. factors (PDGF-AB, PDGF-AA, PDGF-BB), IGF-1, brain- Te next step of preparation consists of the release of derived growth factor (BDNF), epidermal growth factor the growth factors from the platelets. (EGF), VEGF, bFGF and hepatocyte growth factor (HGF) that are suitable to sustain growth of a wide range of cell • Repeated freeze/thaw cycles: the commonest and types [22, 23]. easiest to implement technique consists of cycling Finally, two diferent types of hPL (autologous and allo- (one to fve times) between freezing at −30 or −80 °C geneic), related to the source, were proposed as suitable and thawing at 37 °C to induce fragmentation of the FBS alternatives. Te risk of contamination and adverse platelets [19, 23]. immune reactions is lower in autologous rather than allo- • Direct platelet activation: adding a calcium salt geneic material, but the volume of hPL that can be pro- (commonly CaCl­ 2) which activates the endogenous duced from autologous blood is not sufcient for clinical thrombin cascade and leads to platelets lysis. Alter- use. Autologous hPL also manifests a challenge due to a natively, human or recombinant thrombin can also lack of standardization, related to donor patient’s hetero- be used to induce lysis [31, 32]. geneity which may lead to variations in biological efec- • Sonifcation alone or in combination with freeze/ tiveness [24–26]. thaw cycles: up to 30 min at a frequency of 20 kHz In contrast, derivation of allogeneic hPL can be autom- releases platelet granules rapidly [33, 34]. atized, and standardized in terms of tests and contents, • Solvent/detergent (S/D) treatment: it induces both which means that it could be a good candidate to substi- platelet lysis and inactivates lipid-enveloped virus tute FBS in translational regenerative medicine [27]. [27].

Preparation techniques of hPL A single unit of hPL is obtained by mixing lysates of Te methods applied to prepare hPL could possi- diferent origin. A further centrifugation step is needed bly change its biochemical and functional proper- to deplete the hPL of platelet fragments, then it can be ties, although if and how has not been evaluated yet in stored at − 30 to − 80 °C until use [2]. depth. Pooled allogenic platelets can be obtained from Rauch et al. stated that hPL can be conserved at − 20 °C (e.g. sourced from transfusion banks) by for at least 5 months, maintaining the same concentration three diferent separation protocols: bufy coat (BC), of growth factors, especially EGF [26]. Moreover, hPL platelet-rich plasma (PRP) or plateletpheresis technol- can be stored at 4 °C for 4 weeks without changing its ogy [27]. In the frst technique the whole blood sample efcacy [31], while other authors recently have reported a is frstly centrifuged (3000g for 5 min at 21–22 °C with a longer time period (2 years) over which it maintained its validated acceleration and deceleration curve) to separate properties, if stored at − 80 °C [35]. cell components (bufy coat) from cell-free plasma which forms the top layer of the suspension. Ten the bufy The aim coat of four units (450–525 ml per unit) are mixed with Our aim is to critically appraise and summarize the state a specifc amount of plasma, followed by a second cen- of art in the published literature on how hPL supports trifugation and fltration steps (2μm pore size) in order to in vitro cell culture of hMSCs in comparison to FBS. We remove leukocytes [2, 28]. In the second method (PRP) collated the currently available evidence and envisage four or fve whole blood units are pooled and centrifuged that this will be a helpful guidance in future experimental Guiotto et al. J Transl Med (2020) 18:351 Page 4 of 14

studies, related to the use of hPL and on its use as a sup- MSCs not sourced from human material and papers plement for translational applications. where FBS/FCS controls were lacking. All publications were screened manually, and the data extracted accord- Materials and methods ing to predetermined criteria. Te fow chart of article In February 2019 (repeated in June 2019), a systematic selection follows the Preferred Reporting Items for Sys- review of the entire Web of Science (https​://clari​vate. tematic Reviews and Meta-Analyses (PRISMA) state- com/webof​scien​cegro​up/solut​ions/web-of-scien​ce/), ment (Fig. 1) [36]. Medline (https​://www.nlm.nih.gov/bsd/medli​ne.html) Apart from bibliometric information, the follow- and PubMed (https​://pubme​d.ncbi.nlm.nih.gov) data- ing data was extracted and tabulated for each article base was performed with the following search terms: (Table 1): source of MSCs, concentration of FBS, con- (mesenchymal stem cells) AND (fetal* bovine serum OR centration of hPL, method of platelet extraction, hPL fetal* bovine calf) AND (human platelet lysate). Secondly source (autologous/allogeneic), procedure to create ADSCs AND (FBS OR FBC) AND hPL. Finally, bibliog- platelet lysate, feeding schedule, growth index (prolif- raphy references were analysed and included if pertinent. eration rate, cell count, cell population doubling time All article types, except reviews, that were published or generation time or CFU-U number or cell viability), in English between 2005 and 2019 were considered cell morphology, immunophenotype, MSCs diferentia- without restrictions. Other exclusion criteria were tion potential.

Records idenfied through database searching (n = 271) Idenficaon

Records a er non-pernent abstract removed (n = 91) Screening

Records screened Records excluded (n = 91) (n = 35) Eligibility

Studies included in qualitave synthesis

(n = 56)

Included

Fig. 1 PRISMA fow chart selection process Guiotto et al. J Transl Med (2020) 18:351 Page 5 of 14 CD 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 2 1 1 2 2 1 1 1 3 1 1 1 3 3 1 P.I. 2 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 MO 2 1 1 3 1 1 1 2 2 1 1 3 3 3 1 1 3 4 2 3 1 2 2 1 3 3 2 1 1 2 3 3 T. Feeding T. 3–4 days 3–4 days 3–4 days N.M. 3–4 days 3–4 days 3–4 days 3–4 days 3–4 days 1 day 3–4 days N.M. 3–4 days 3–4 days N.M. N.M. 3–4 days 3–4 days 3–4 days 3–4 days 3–4 days 3–4 days N.M. 3–4 days 3–4 days N.M. N.M. 3–4 days 3–4 days 3–4 days N.M. 3–4 days Dif. Dif. Line 1 3 1 1 0 1 A − /B + 0 1 B 0 A − /B + 1 0 1 1 1 0 A − /B + 1 1 3 0 1 1 1 1 1 1 1 A − /B + A Filter N.M 0.22/0.1 µm N.M N.M 0.22 µm 0.45/0.22 µm 0.22 µm 0.2 µm N.M 0.2 µm N.M N.M 70 µm N.M 0.2 µm N.M N.M N.M 0.22 µm N.M N.M N.M N.M N.M N.M 0.2 µm N.M 0.2 µm 0.2 µm N.M 0.45 µm 0.22 µm UV, + UV, FT Ca, + Ca, FT Type of lysis/ Type Inactivation N.M. SD N.M. N.M. FT FT SD SD FT FT N.M. N.M. Ca, FT Ca, Ca FT N.M. UV, FT UV, N.M. FT N.M. N.M. FT FT N.M. FT FT N.M. UV FT FT Thr, FT Thr, Ca, FT Ca, hPL manufacturing I Aph I I BC BC Aph Aph N.M. N.M. I I Aph PRP Aph I BC I BC I PRP PRP Aph I PRP Aph N.M. BC PRP BC BC PRP + hPlasma bFGF, hPL, PR-hPL bFGF, + 7.50% (SDPL), 6.72% GBHPL 5% PR-SRGF hPLSP % Additive 10% FBS, hPL, HS FBS, hPL, i-hPL FBS 10%, xeno free media free FBS 10%, xeno 10% FBS, 5% hPL FBS 10%, 5% hPL FBS 10%, hPL 5% 10% FBS, 6.55%(FTPL), 6.50%(GBPL), 10% FBS, 6.55%(FTPL), 6.50%(GBPL), 20% FBS, 10% HPGF-C18 20% FBS, 10%, 5% hPL 10% FBS, hPL, hPL 10% FBS, 5% hPL FBS 10%, hPL 10% 10% FBS, 10%, 7.5%, 5% hPL, 7.5% 10% FBS, 10% PL-S, 10% PL-P 10% FBS, PL-S, 10%FBS, 10% hPL FBS, hPL, hS 2.5%, 5%, 10% 2-15% FBS 10% FBS, 10%, 5% hPL FBS 10%, hPL 5% 10% FBS, PL 10% FBS, 2%, 5%, 7%, PL 10% FBS, 10%, 5% UCB-PL, PB-PL 10% FBS, PHPL, hPL 10% FBS, 5% PLTMax, 5% hPLS, 10% FBS, 5% PLTMax, 10% FCS, 5% PPP, hPL 10% FCS, 5% PPP, 10% FBS, 5% hPL 10% FBS, 5%, 7.5%, XcytePLUS-hPL 10% FBS, hPL, iHPL 10% FBS, hPL 10% FBS, 5% hPLp, 5% hPLn, 10% huS 10% FBS, 5% hPLp, 10%, 7.5%, 5%, 2.5% FBS, pHPL, tPRP 10%FBS, 10% hPLp, 10% hPLs 10%FBS, 10% hPLp, Type of cell Type hUMC-MSCs BM-MSCs ASC-MSCs ASC-MSCs ASC-MSCs BM-MSCs hUMC-MSCs BM-MSCs hUMC-MSCs ASC-MSCs ASC-MSCs ASC-MSCs BM-MSCs BM-MSCs BM-MSCs hC-MSCs BM-MSCs ASC-MSCs ASC-MSCs BM-MSCs hUMC-MSCs hUMC-MSCs hC-MSCs ASC-MSCs and BMSCs BMSCs, ASCs, UC-MSCs, T-lymph UC-MSCs, ASCs, BMSCs, ASC-MSCs and hF hUMC-MSCs BM-MSCs ASC-MSCs ASC-MSCs and hF ASC-MSCs and BMSC MSCs Selected publications between 2005 and 2019 in Web of Science database according to the following search terms: (mesenchymal stem cells) AND (fetal AND (fetal cells) stem (mesenchymal terms: search to the following according of Science database Selected and 2019 between 2005 publications in Web 1 Table checking bibliography calf) bovine and complete OR fetal serum bovine its acronyms lysate), AND (human platelet Author al. 2019) (Kong et al. al. 2019) et al. (Barro al. 2018) et al. (Lensch al. 2018) et al. (Haack-Sørensen al. 2019) (Dessels et al. al. 2018) (Becherucci et al. al. 2019) et al. (Chen al. 2018) (Ren et al. (Kandoi 2018) et al. al. 2017) et al. (Phetfong al. 2017) et al. (Søndergaard al. 2017) (Mangum et al. al. 2017) et al. (Bernardi al. 2017) et al. (Pierce al. 2017) et al. (Fernandez-Rebollo al. 2017) et al. (Matthyssen (Viau 2017) et al. al. 2016) (Riis et al. (Escobar and Chaparro, 2016) (Escobar and Chaparro, al. 2016) (Heathman et al. al. 2016) (Mohammadi et al. al. 2017) (Shirzad et al. al. 2016) (Suri et al. (Juhl 2016) et al. al. 2015) (Muraglia et al. (Wagner 2015) et al. al. 2015) (Riordan et al. al. 2014) et al. (Castiglia al. 2013) Kølle et al. Trojahn (Witzeneder 2013) et al. (Kinzebach and Bieback, 2013) al. 2013) (Mojica-Henshaw et al. Guiotto et al. J Transl Med (2020) 18:351 Page 6 of 14 CD 1 3 1 1 1 1 3 1 1 1 1 1 1 1 1 1 2 1 1 3 1 1 1 1 P.I. 1 1 1 1 1 2 1 1 1 1 1 3 2 1 1 2 1 1 1 1 1 1 1 1 MO 3 3 1 1 1 3 3 1 2 3 1 2 1 1 3 2 1 1 1 1 1 2 2 3 T. Feeding T. 3–4 days N.M. N.M. 3–4 days N.M. 3–4 days N.M. 3–4 days 3–4 days N.M. 3–4 days 3–4 days N.M. 3–4 days 3–4 days 3–4 days 1 day 3–4 days 3–4 days 3–4 days 3–4 days 3–4 days 3–4 days 3–4 days Dif. Dif. Line 1 0 1 AB − 3 1 0 0 C 1 1 N A − /B + 1 0 1 1 1 1 1 1 1 1 A Filter 70 µm N.M N.M N.M N.M 0.45/0.22 µm 0.22 µm N.M 0.2 µm 70 µm 0.40 µm N.M 0.22 µm N.M N.M 0.2 µm 0.22 µm 0.2 µm N.M N.M 0.22 µm N.M 0.22 µm N.M Type of lysis/ Type Inactivation Son, FT FT FT FT FT UV, FT UV, FT FT SD FT FT FT FT FT FT FT FT Thr, FT Thr, FT FT FT FT FT FT hPL manufacturing PRP N.M. PRP Aph N.M. BC BC, Aph BC N.M. N.M. PRP PRP Aph N.M. Aph BC, Aph N.M. PRP N.M. PRP PRP PRP PRP Aph 5% hPL, 10% + PLT 5% hPL % Additive 10% FBS, 10% PLT-FT-Ly, 7.5%, 5%, 2.5% 10% FBS, PLT-FT-Ly, 10/0, 7.5/2.5, 5/5, 2.5/7.5, 0/10% FBS, hPL 16% FBS, 2%, 5%, 10% hPL 10%FBS, 10% FBS 10% FBS, hPL 20% FBS, 10% hPL 10% FBS, 2.5, 5, pHPL 10% FBS, 5% hPL 10% FBS, hPL FBS 10%, hPL 5% 10% FBS, hPL 10% FBS, 5% hPL 10% FBS, 7.5% hPL 10% FBS, hPL 10% FBS, hPL 10%FBS, 10% hPL 10% FBS, 2.5%, hPL 10% FBS, 10% HS, 10% tPRP, 10% pHPL 10% FBS, HS, tPRP, 10% FBS, 5% hPL 10% FBS, 5% hPL 10% FBS, hPL 10% FBS, 5% hPL 10% FBS, hPL 10% FBS, 5% hPL Type of cell Type BM-MSCs ASC-MSCs BM-MSCs BM-MSCs BM-MSCs BM-MSCs hUMC-MSCs ASC-MSCs ASC-MSCs ASC-MSCs and BMSCs DPSCs BM-MSCs BM-MSCs ASC-MSCs BM-MSCs BM-MSCs ASC-MSCs BM-MSCs BM-MSCs BM-MSCs BM-MSCs BM-MSCs hUMC-MSCs BM-MSCs (continued) 1 Table Author (3) (2); Not mentioned : Higher in hPL (1); No diference (P.I.) rate Proliferation (3) or < (2); Not mentioned = (1); No expression CD >/ : Expression Immunophenotype (CD) shape (4) (3); Diferent (2); Not mentioned spindle shape (1); No diference : Smaller elongated Morphology (MO) adipogenic and − ); Low (AB adipogenic and osteogenic (1); High High (B); High(C); trilineage Low osteogenic (3); High chondrogenic adipogenic (A); (0); No diference Line) : Not mentioned lineage (Dif. Diferentiation (N) diferentiation − /B + ); Neural (A high osteogenic (N.M.) Not mentioned and/or Bufy Coat; Apheresis hPL manufacturing (Aph); : Industrial (BC); plasma (PRP); Apheresis (I); Bufy coat Platelet-rich activation (Thr)Thrombin Irradiation (UV); (FT); and/or FT; inactivation + irradiation Sonifcation addition (Ca); (Son); inactivation Solvent/detergent (SD); Calcium : Freeze/thaw Solvent/detergent lysis of platelet Type FT); FT); addition and/or FT (SD + Ca, inactivation + Ca and/or FT (SD + UV, FT); Solvent/detergent FT); addition and/or FT (Ca, Calcium Irradiation and/or FT (UV, (N.M.) Not mentioned and/or human (ACS-MSCs) derived-MSCs MSCs (hC-MSCs); Adipose stromal Human corneal (ACS-MSCs); derived-MSCs (BM-MSCs); Adipose (hUMC-MSCs); Bone marrow-MSCs : Human umbilical cord-MSCs of cells Type histiocytic hUMC-MSCs, lymphoma U-937, AT-MSCs, (MSCs); BM-MSCs, (BM-MSCs); Not specify the source and/or Bone marrow-MSCs (ACS-MSCs) derived-MSCs skin Pulp-MSCs (DPSCs); Adipose (hF); Dental fbroblast CD4/CD8 (Others) T-lymphocyte human chondrocyte, al. 2013) et al. (Bernardi (Shanskii 2013) et al. al. 2013) (Grifths et al. (Ben Azouna 2012) et al. al. 2012) (Gottipamula et al. al. 2012) et al. (Fekete (Schallmoser and Strunk, 2013) al. 2012) (Naaijkens et al. al. 2011) (Shih et al. al. 2011) et al. (Crespo-Diaz al. 2011) et al. (Govindasamy al. 2011) (Shichinohe et al. (Xia 2011) et al. al. 2011) et al. (Cholewa al. 2011) et al. (Abdelrazik al. 2010) (Horn et al. al. 2009) (Blande et al. al. 2009) (Bieback et al. al. 2009) et al. (Prins (Zaky 2008) et al. al. 2007) (Schallmoser et al. al. 2007) et al. (Capelli al. 2007) (Reinisch et al. al. 2005) (Doucet et al. Guiotto et al. J Transl Med (2020) 18:351 Page 7 of 14

Table 2 Proliferation rate Table 4 Diferentiation lineage Proliferation rate (P.I.) N % Diferentiation lineage (Dif. Line) N %

Higher in hPL (1) 47 83.93 Not mentioned (0) 10 17.86 No diference (2) 8 14.29 No diference (1) 32 57.14 Not mentioned (3) 1 1.79 High trilineage (3) 3 5.36 High Adipogenic (A) 2 3.57 High osteogenic (B) 1 1.79 High chondrogenic (C) 1 1.79 Table 3 Immunophenotype Low adipogenic and osteogenic (AB ) 1 1.79 − Immunophenotype (CD) N % Low adipogenic and high osteogenic (A /B ) 5 8.93 − + Expression CD >/ (1) 44 78.57 Neural diferentiation (N) 1 1.79 = No expression or < (2) 6 10.71 Not mentioned (3) 6 10.71 Table 5 Morphology Morphology (MO) N % Results Smaller elongated spindle shape (1) 25 44.64 Te initial search process found 271 papers, which was No diference (2) 13 23.21 reduced to a fnal selection of 56 articles, excluding Not mentioned (3) 17 30.36 reviews, studies published before 2005, articles where the Diferent shape (4) 1 1.79 FBS/FCS control were missing or when MSCs came from other sources than human tissue (Table 1). Te major- ity of the papers (83.9%) reported a higher proliferation index for the hMSCs cultured with hPL compared to hMSCs were mainly sourced from bone marrow (BM- those cultured with FBS (without any distinction based MSCs, 42.9%) or adipose tissue (AT-MSCs, 25%). In on the extraction or lysate preparation methodology). In 5.4% of the studies hMSCs isolated from both of these the remaining (14.3%) no diferences were mentioned, sources were used. Alternative sources (globally 23.2%) while only one case lacked this information (Table 2). Te such as dental pulp stem cells (DPSCs), corneal stromal evaluation of immunophenotype showed that in both cells (hCMSCs) and umbilical cord mesenchymal cells FBS and hPL culture, the cells expressed similarly surface (hUMCs) were also tested without relevant statisti- markers CD73, CD90, and CD105, in about 78.6% of the cal diference between hPL and FBS additive medium, papers. except for proliferation where, similarly to BM and AT, A lower or lack of expression of these three CD mark- hPL accretes it. In one case the origin of the hMSCs ers was reported in 10.7% of the studies, while in another was not mentioned (Table 6). 10.7% it was not mentioned (Table 3). Te diferentia- PRP (26.8%), plateletpheresis (19.6%) and bufy coat tion capacity along the osteocyte, adipocyte, and chon- (16.1%) were the most prevalent manufacturing meth- drocyte lineage did not show any signifcant diference in ods. Te remaining 33.9% of papers applied an indus- the majority of the studies (57.1%). In 10.7% of reports, trial preparation or did not specify the technique or a lower adipogenic potential was highlighted. Just a few origin of the hPL (Table 7). Te platelets were lysed by papers showed a higher potential for hPL-hMSCs to dif- repeated freeze/thaw cycles in 57.1% of the reports, ferentiate into only the osteocyte (10.7%), all three line- direct platelet activation by adding thrombin was used ages (5.4%) or adipocyte (3.6%) or chondrogenic (1.8%) in 3.6%, solvent/detergent (S/D) treatment in 3.6%, line. In one case neurogenic diferentiation was obtained ­CaCl2 addition (1.8%) and sonifcation in 1 case (1.8%). with a specifc protocol. In the remaining 17.9% of the Sterilisation of hPL by irradiation of the platelet lysate papers, the diferentiation potential was not tested was described in 7.1% of the cases. A combination of (Table 4). these diferent techniques with UV treatment was Analyzing cell morphology, the hMSCs phenotype described in 2 out of 56 cases (3.6%) with freeze/thaw with hPL was more often (44.64%) spindle-shaped, elon- (F/T) and one associated with solvent/detergent (S/D) gated and smaller compared to cells cultured in FBS, treatment (1.8%). Finally, a S/D treatment plus cal- while in 23.2% no signifcant morphological diferences cium addition followed by F/T was mentioned in one were found. In 37.9% of articles cell morphology was not case (1.8%). Sterilisation methods were not reported in described. (Table 5). 19.6% of publications (Table 8). Guiotto et al. J Transl Med (2020) 18:351 Page 8 of 14

Table 6 Type of cell Type of cell N %

Human umbilical cord-MSCs (hUMC-MSCs) 8 14.29 Bone marrow-MSCs (BM-MSCs) 24 42.86 Adipose derived-MSCs (ACS-MSCs) 14 25.00 Human corneal stromal MSCs (hC-MSCs) 2 3.57 Adipose derived-MSCs (ACS-MSCs) and/or human skin fbroblast (hF) 2 3.57 Dental Pulp-MSCs (DPSCs) 1 1.79 Adipose derived-MSCs (ACS-MSCs) and/or Bone marrow-MSCs (BM-MSCs) 3 5.36 Not specify the source (MSCs) 1 1.79 BM-MSCs, ASC-MSCs, hUMC-MSCs, histiocytic lymphoma U-937, human chondrocyte, T-lymphocyte CD4/ 1 1.79 CD8 (Others)

Table 7 hPL manufacturing Table 9 Filter hPL manufacturing N % Filter N %

Industrial (I) 9 16.07 0.2 µm 18 32.14 Bufy coat (BC) 9 16.07 0.4 µm 2 3.57 Platelet-rich plasma (PRP) 15 26.79 70 µm 3 5.36 Apheresis (Aph) 11 19.64 0.2/0.1 µm 1 1.79 Apheresis and/or Bufy Coat 2 3.57 0.4/0.2 µm 2 3.57 Not mentioned (N.M.) 10 17.86 N.M. 30 53.57

Table 8 Type of platelet lysis In Table 10 the main growth factors and protein com- Type of platelet lysis N % position of the hPL are listed. Compared to FBS the con- centration of PDGF-AA, -AB, -BB, TGF-Beta, VEGF and Freeze/thaw (FT) 32 57.14 IGF-1 is higher in hPL, while the total protein and albu- Solvent/detergent inactivation (SD) 2 3.57 min values are respectively lower or the same. Sonifcation (Son) 1 1.79 However, in the majority of the studies the analysis of Calcium addition (Ca) 1 1.79 the growth factors composition was limited to compare Thrombin activation (Thr) and/or FT 2 3.57 the diferent type of hPL lysate preparations, instead of Irradiation (UV) 1 1.79 analysing the variation between FBS and hPL. No sig- solvent/detergent inactivation irradiation and/ 1 1.79 nifcant variation in GF composition was discovered that or FT (SD UV, FT) + + related to platelet lysate manufacturing methods. solvent/detergent inactivation Ca addition 1 1.79 and/or FT (SD Ca, FT) + + Calcium addition and/or FT (Ca, FT) 2 3.57 Discussion Irradiation and/or FT (UV, FT) 2 3.57 Currently, there is no consensus in the literature regard- Not mentioned (N.M.) 11 19.64 ing the most appropriate medium additives for in vitro expansion of hMSCs in a clinical setting. According to our selection criteria, all included publications analyzed Overall, 54%of the studies did not mention the type hMSCs morphology, proliferation, immunophenotype of flter used, while 32% applied a 0.2 µm, 2 out of 56 and mesodermal diferentiation potential, allowing the papers a 0.4 µm, 3 articles a 70 µm or a combination of efects of hPL or FBS supplement on these parameters to 0.4/0.2 µm (2) and 0.2/0.1 µm (1) (Table 9). be compared. Most studies, assessed here, used hMSCs Te heterogeneity of the protocols used to prepare derived from bone marrow or adipose tissue. hPL further increased, with the inclusion of throm- Tis lack of consensus can be explained by the intrin- bin or heparin in the suspension or additional extrac- sic variability and complexity of both FBS and hPL: both tion conditions (centrifugation and additional fltration their composition is not completely defned in terms steps). of protein, cytokine and elementary components and, moreover, hPL manufacturing is not a standardized Guiotto et al. J Transl Med (2020) 18:351 Page 9 of 14 Albumin Albumin (mg/ml) 41.00 17.00 16.00 16.12 42.50 Protein Protein Tot (mg/ ml) 58.32 26.85 26.27 56.00 56.00 56.00 56.00 54.00 90.46 49.14 61.20 7.70 66.60 58.64 26.00 Beta- NFG 0.00 HGF (ng/ ml) 0.13 0.25 0.24 0.12 0.12 0.12 0.12 0.5 0.00 IGF-1 (ng/ ml) 80.00 80.00 35.17 17.50 17.50 17.50 17.50 0.00 0.11 32.55 32.55 0.00 501.62 325.50 33.14 4.42 85.98 108.70 130.00 BDNF (ng/ ml) 100.00 80.00 25.73 20.00 20.00 20.00 20.00 5.5 0.79 EGF (ng/ ml) 3.80 5.90 4.70 0.85 0.85 0.85 0.85 0.5 0.00 0.00 3.40 1.48 1.48 0.42 0.20 0.27 0.01 0.01 0.01 4.40 3.60 20.00 bFGF (ng/ml) 0.40 0.42 0.35 0.55 0.55 0.55 0.55 5.5 0.00 0.00 0.20 0.15 0.15 0.30 0.06 0.06 0.36 0.17 0.00 0.19 0.16 0.50 0.20 VEGF (ng/ml) 0.17 0.40 0.31 0.40 0.40 0.40 0.40 0.75 0.00 0.00 0.80 0.65 0.65 0.00 0.90 1.20 0.15 0.33 0.45 TGF- Beta (ng/ml) 32.00 33.00 35.17 75.00 75.00 75.00 75.00 5.5 0.05 0.04 55.20 55.20 0.15 56.04 29.31 250.00 210.00 32.74 54.86 4.89 16.30 139.03 125.00 2.85 PDGF-BB (ng/mL) 11 11 11 11 5.24 0.01 0.01 10.70 0.42 18.11 22.14 2.38 13.90 8.70 8.20 0.14 PDGF-AB PDGF-AB (ng/ml) 80.00 125.00 8.16 45.00 45.00 45.00 45.00 17.84 0.03 0.14 28.78 28.78 577.97 391.62 50.00 43.80 3.98 571.73 PDGF-AA PDGF-AA (ng/ml) 4.57 0.00 0.01 21.00 13.70 6.91 239.41 PDGF (ng/ml) 70.00 40.00 10.00 thaw thaw -196/4 °C thaw thaw -80/4 °C Type Type of Medium hPL I-hPL DVI-hPL SDPL FTPL GBPL GBHPL hPL hPL PR-SRGF hPL PR-hPL hPL hPL UCB-PL PB-PL hPL PhPL hPL p hPL n huS hPLFreeze/ hPL Freeze/ hPL PL-PCC hPL hPL Estimated? Y Y N N N N N N N N N N N N N N Y Y N N N N N N N N N Year 2019 2019 2019 2018 2018 2018 2018 2017 2017 2017 2017 2017 2017 2016 2016 2016 2016 2016 2013 2013 2013 2013 2013 2013 2011 2011 2011 Composition of human platelet lysate reported in literature: growth factors and proteins growth reported in literature: lysate of human platelet Composition - - - - zeneder zeneder zeneder Hen - shaw Hen - shaw Diaz che 10 Table First author Barro Barro Barro Chen Chen Chen Chen Mangum Bernardi M Bernardi Bernardi M Bernardi Pierce Viau Viau Escobar Shirzad Shirzad Suri Suri Wit Wit Wit Mojica- Mojica- Shanskii Fekete Crespo- Shichino Guiotto et al. J Transl Med (2020) 18:351 Page 10 of 14 Albumin Albumin (mg/ml) 26.52 34.45 34.45 Protein Protein Tot (mg/ ml) 49.95 58.64 58.64 Beta- NFG 0.00 0.00 0.00 0.00 0.00 HGF (ng/ ml) 0.13 0.00 0.00 1.72 2.55 IGF-1 (ng/ ml) 0.00 106.67 107.41 78.81 64.10 72.82 68.46 BDNF (ng/ ml) 35.82 0.00 0.00 0.03 0.05 EGF (ng/ ml) 0.19 2.77 bFGF (ng/ml) 0.04 0.06 0.09 0.00 0.23 0.00 0.00 VEGF (ng/ml) 0.00 0.40 0.00 0.00 TGF- Beta (ng/ml) 5.15 50.84 0.51 57.24 0.85 1.27 6.21 2.78 1.73 3.84 PDGF-BB (ng/mL) 7.50 0.00 0.14 0.07 0.00 0.00 PDGF-AB PDGF-AB (ng/ml) 0.00 40.46 30.89 0.24 99.06 0.03 0.00 0.01 PDGF-AA PDGF-AA (ng/ml) 7.42 36.63 0.14 0.14 PDGF (ng/ml) 40.00 cultures cultures Type Type of Medium hPL hPL hPL PPP FBS FBS FCS FBS-BMSCs hPL-BMSCs hPL-BMSCs Estimated? N N N N N N N N N Year 2011 2007 2005 2005 2013 2011 2005 2011 2011 (continued) - - - moser Values che che Values che 10 Table First author Xia Schall - Doucet Doucet Shanskii Mean Shichino Doucet Shichino Mean Shichino Guiotto et al. J Transl Med (2020) 18:351 Page 11 of 14

process. All these factors increase the challenge to judge how they afect hMSCs grown in hPL. Because some the respective usefulness of these media supplements. authors compared FBS with industrial hPL products, Despite these diferences, the majority of the authors which were prepared using undisclosed methods of isola- agreed that media supplemented with hPL support a tion and lysis, we could not draw any conclusion of the higher proliferation index compared to FBS supple- infuence of hPL isolation and preparation methods on mented ones or other, serum-free media [3, 9, 10, 13, 15– cells. 17, 19, 24, 31, 33, 35, 37–65]. Te release of platelet content to create hPL can be A conceivable explanation of this data has to be found achieved by a variety of methods: freeze and thaw, acti- in the composition of hPL; platelet alfa-granules contain vation by either thrombin or calcium, solvent/detergent coagulation factors, adhesion molecules, protease inhibi- treatment and sonifcation. In summary of the assembled tors, bFGF, EGF, HGF, VEGF, IGF-1, TGF-ß, PDGF-AA, data from the publications reviewed here, the technique PDGF-AB, PDGF-BB and a variety of cytokines and by which lysis was achieved seems to have no signifcant chemokines [66, 67]. Teir respective role in conveying infuence on MSCs growth, proliferation or diferentia- the increase in cell growth and proliferation rates and tion potential [13, 20, 31, 33, 37, 40, 45, 54, 66, 69]. thus cell expansion has not yet been completely resolved. Te risk of biological contamination of hPL could One probable cause could be the known efect of some be minimized by adapting manufacturing methods to of these growth factors to increase cell proliferation (par- include additional steps such as detergent, UV irradia- ticularly PDFG-BB and bFGF) [67]. tion, or irradiation in combination with psoralen. Shih Specifcally, three studies compared the growth factor illustrated how inactivating hPL by solvent/detergent concentration in both FBS and hPL showing that in FBS treatment did not afect hMSCs proliferation, lineage dif- the following growth factors are present in lower quan- ferentiation capability and CD marker expression com- tity than in hPL (mean values respectively for FBS and pared to either FBS or hPL control [70]. In the same way hPL in ng/mL): PDGF-AA (0.14, 36.63), PDGF-AB (0.01, photoactivation with UV light or psoralen did not alter 99.06), PDGF-BB (0.07, 7.50), TGF-ß (2.78, 57.24), bFGF BM-MSCs growth or CD markers of stemness [71, 72]. (0.0, 0.23), VEGF (0.0, 0.40), (IGF-1) (68.46, 78.81) and Further studies are needed as the diferent techniques (BDNF) (0.0, 35.82) (Table 2) [19, 55, 68]. used for hPL generation could conceivably interfere with In about half of the publications the authors specifcally its composition, for instance, the quality and quantity of mentioned that MSCs expanded in hPL showed a dif- growth factors and cytokines. ferent morphology, compared to those grown with FBS; Despite the promising data which showed that hPL can they were smaller, more elongated and more often spin- be an excellent alternative to FBS, open questions remain dle shaped. In the remaining publications, the authors did regarding quality and production. At this time, accord- not investigate or did not mention any change in cellular ing to our fndings, not enough information is avail- morphology. Only one publications reported hMSCs to able due to a lack of universally accepted international be larger when cultured with hPL [46]. guidelines for hPL preparation and analysis. Recently, in In the vast majority of reports the ability of hMSCs response to similar results reported elsewhere, a collabo- to be diferentiated along pre-determined lineages was ration between the American Association of Blood Banks not afected by being cultured using hPL: adipogenic, (AABB) and the International Society of Cell Terapy osteogenic and chondrogenic diferentiation were dem- was initiated to address hPL quality control and stand- onstrated with no relevant diference in efectiveness or ardize hPL manufacturing [30, 73]. potential to the same hMSCs cultured with FBS. In three cases MSCs, expanded in hPL, were more efectively Limitations diferentiated along osteogenic, chondrogenic and adi- Te data, in the selected literature revised, showed a large pogenic routes [37, 49, 58]; others reported a diference variety in terms of defnitions e.g. the proliferation index, only for osteogenic [17, 40, 42, 47, 54] or adipogenic dif- as we named it, incorporates any measurements related ferentiation respectively [19, 31]. Only one publication to cell proliferation such as repeated cell counts, popu- reported that FBS supported osteogenic and adipogenic lation doubling time (PDT), cumulative PDT, generation diferentiation to a higher degree [57]. A single study time, number of colony forming unit fbroblast (CFU- investigated neural diferentiation of BM-MSCs (growth F); the methods and values varied considerably between media containing 10% FCS and 5% hPL respectively), studies, made the comparison difcult and sometimes reporting no signifcant diferences in terms of prolifera- even impossible to arrive at any meaningful conclusion. tion and diferentiation potential [68]. Moreover, the methodology of hPL manufacturing, Te BC and PRP techniques, that were applied to as previously highlighted, encompassed a wide range of obtain the pooled platelets, were not distinguished in parameters, starting with the heterogenicity of blood Guiotto et al. J Transl Med (2020) 18:351 Page 12 of 14

sample management, the extraction protocols, such as It is necessary to defne a minimal concentration for BC or PRP, and the multiple platelet lysis strategies. Fur- the main components due to be sure that the fnal prod- thermore, almost each publication mentioned additional uct can support MSCs adequately, independently of the steps that deviated from the generalised protocol e.g. diferent manufacturing methods or lysis or pathogen additional centrifugation, fltration, thrombin or heparin inactivation. addition, suspension in diferent intermediate solution or Reaching the minimum content requirements, to irradiation steps. All these aspects somewhat limited our reduce costs and preserve the limited sources of PL, a ability to combine the data in a meaningful way. blended mixture with serum free and lysate could be For future works we strongly recommend to the speculated to obtain an ideal growth cell environment. authors: (A) include FBS controls (lead to the exclusion of Studies which compared diferent PL extraction with 11 publications after a further selection of 271 records); same lysis process or the variety of lysis techniques from (B) provide a detailed preparation methodology for hPL; the same isolation strategy, are needed to understand the (C) measure the growth factor, cytokine and micronu- most efcient combination in terms of GFs and protein trient content; (D) include a clear characterization of content. Tis could address to a standardization of the hMSCs phenotype including morphology, CD marker methods to obtain a more comparable hPL. profle and trilineage diferentiation capability. Acknowledgements Finally, the proliferation index must be comparable: The authors are grateful to the FONDATION LEENAARDS for the grant which researchers use a wide range of methods such as repeat supports our research projects. cell counts, population doubling time, cumulative popu- Authors’ contributions lation doubling time, generation time, MTT or Alamar PGS and MOR conceived the original idea and supervised the project. MG blue and number of colony forming unit-fbroblasts analyzed the data and wrote the manuscript. All authors discussed the results and contributed to the fnal manuscript. All authors read and approved the (CFU-F) to evaluate the cell growth, which reduces the fnal manuscript. ability to compare the respective data. Funding LEENAARDS FONDATION is supporting fnancially our research works.

Conclusion and future perspectives Availability of data and materials In the literature reviewed, no consensus was expressed Yes. in terms of the source of the medium supplement (FBS/ Ethics approval and consent to participate hPL), neither a standard procedure of hPL manufactur- Not applicable. ing clearly emerged. Te quality of the fnal product, in Consent for publication terms of diferent impact on hMSCs biology, may be sig- Not applicable. nifcantly infuenced by the production methods utilized. Despite this heterogeneity in hPL manufacturing, the Competing interests The authors declare that they have no competing interests. majority of the publications agreed that hPL was at least as efective as FBS with regards to maintaining hMSCs Author details 1 proliferation, immunophenotype and diferentiation Department of Plastic, Reconstructive and Hand Surgery, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland. 2 Centre for the Cellular capacity. Microenvironment, University of Glasgow, Glasgow, UK. 3 Canniesburn Plastic According to the international society of cellular ther- Surgery Unit, Glasgow Royal Infrmary, Glasgow, UK. apy (ISCT) and the international fat applied technology Received: 5 February 2020 Accepted: 20 August 2020 society (IFATS) which have demanded specifc guidelines for the characterization and selection of ADSCs in regen- erative medicine, hPL could be considered an alternative to the use of FBS in hMSCs culture—especially with a References view to their clinical use. 1. Robey P. “Mesenchymal stem cells”: fact or fction, and implications in Based on our review we envisage that hPL research their therapeutic use. F1000Res. 2017;6:F1000. 2. Shih DT, Burnouf T. Preparation, quality criteria, and properties of human should address a standardization of hPL production pro- blood platelet lysate supplements for ex vivo stem cell expansion. N cess. hPL should ideally come from a limit number of Biotechnol. 2015;32:199–211. pooled PL (platelet) units in order to minimize the risk 3. Lensch M, Muise A, White L, Badowski M, Harris D. Comparison of syn- thetic media designed for expansion of adipose-derived mesenchymal of patient-to-patient disease transmission but guarantee stromal cells. Biomedicines. 2018;6:54. the highest levels of GFs in the fnal product. Te samples 4. Sotiropoulou PA, Perez SA, Salagianni M, Baxevanis CN, Papamichail M. have to be tested for a standardized set of bacterial, yeast, Cell culture medium composition and translational adult bone marrow- derived stem cell research. Stem Cells. 2006;24:1409–10. fungal and viral contamination and PL donors have to be 5. Perez-Ilzarbe M, Diez-Campelo M, Aranda P, Tabera S, Lopez T, del Canizo excluded in case of a potential anamnesis exposure. C, Merino J, Moreno C, Andreu EJ, Prosper F, Perez-Simon JA. Comparison Guiotto et al. J Transl Med (2020) 18:351 Page 13 of 14

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