Expansion of adipose tissue mesenchymal ... - Wiley Online Library

TRANSFUSION PRACTICE Expansion of adipose tissue mesenchymal stromal progenitors in serum-free medium supplemented with virally inactivated allogeneic human platelet lysate _2915

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Daniel Tzu-Bi Shih, Jung-Cheu Chen, Wan-Yu Chen, Ya-Po Kuo, Chen-Yao Su, and Thierry Burnouf

BACKGROUND: Single-donor or pooled platelet lysates (PL) can substitute for fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) expansion. However, for clinical applications of MSCs, the use of virally inactivated PL would be desirable. Recently, we have developed a solvent/detergent (S/D)-treated human PL preparation (S/D-PL) rich in growth factors. The capacity to use this virally inactivated preparation for MSC expansion needs to be evaluated. STUDY DESIGN AND METHODS: Platelet concentrates were treated by S/D (1% tri-n-butyl phosphate and 1% Triton X-45), extracted by oil, purified by C18 hydrophobic interaction chromatography, and sterile filtered. S/D-PL was compared to FBS as a medium supplement (10% vol/vol) for isolating, maintaining, and expanding adipose tissue–derived MSCs (AT-MSCs). Cell morphology; proliferation kinetics; immunophenotype; differentiation capacity toward the chondrogenic, osteogenic, and osteogenic lineages; and cytokine antibody array were assessed. RESULTS: AT-MSCs had a typical spindle morphology and proliferated in S/D-PL at least as well as in FBS. Immunophenotype at Passage 7 was characteristic of MSCs and similar for both culture conditions. Differentiation capacity into the three lineages was maintained and chondrogenesis was enhanced by S/D-PL. In a 120 human cytokine antibody array analysis, 73 cytokines were detected in S/D-PL, including 22 with a concentration higher than in FBS. CONCLUSION: S/D-PL is an alternative to FBS for AT-MSC maintenance and expansion, does not compromise the differentiation capacity nor the immunophenotype, and may accelerate chondrogenesis. S/D-PL protocols for MSC clinical scale-up may represent a major step toward challenging new use in stem cell therapies.

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esenchymal stromal cells (MSCs) derived from adult tissues of mesodermal origin exhibit various degrees of plasticity in vitro and in vivo, which have been attributed to culture conditions, mesenchymal-to-epithelial transition1 and fusion.2 These populations do not develop into sizable proportions under normal culture conditions, and their isolation and expansion require enriched substrates and culture media. In spite of their presumed existence in vivo as well as in vitro, accumulating evidence indicates that aging decreases the frequency, growth, and differentiation potential of adult MSCs.3-7 MSCs obtained from various sources such as marrow, umbilical cord blood, or adipose tissue can be expanded ex vivo and differentiated into several cell lineages such as osteoblasts, adipocytes, and chondrocytes, as well as hepatocytes and neuronallike cells, therefore generating much interest for their

ABBREVIATIONS: AT-MSC(s) = adipose tissue–derived mesenchymal stromal cell(s); EGF = epidermal growth factor; FGF = fibroblast growth factor; GF(s) = growth factor(s); IGF = insulin-like growth factor; LG = low glucose; MSC(s) = mesenchymal stromal cell(s); PDGF(s) = plateletderived growth factor(s); PL = platelet lysate; PLA = processed lipoaspirate; S/D-PL = solvent/detergent–treated human platelet lysate preparation; TnBP = tri-n-butyl phosphate; VEGF = vascular endothelial growth factor. From the Graduate Institute of Medical Sciences, Taipei Medical University; the Genomic Research Center, Academia Sinica; the Institute of Oral Biology and the Department of Dentistry, National Yang-Ming University; and the College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; and the Human Protein Process Sciences (HPPS), Lille, France. Address reprint requests to: Thierry Burnouf, PhD, Human Protein Process Sciences, 18 Rue Saint-Jacques, 59800 Lille, France; e-mail: [email protected]. Received for publication June 17, 2010; revision received August 19, 2010, and accepted September 2, 2010. doi: 10.1111/j.1537-2995.2010.02915.x TRANSFUSION 2011;51:770-778.

VIRALLY INACTIVATED PLT LYSATE FOR MSC EXPANSION

potential in cell therapy and regenerative medicine.8 Clinical evaluations are under way such as in bone diseases, graft-versus-host disease, engraftment of marrow transplants, or myocardial infarction.9 Several clinical applications have lagged due to technical issues, including the fact that most in vitro MSC isolation and expansion protocols require growth medium supplemented with bovine serum, most often from fetal origin (FBS). FBS is a complex mixture providing essential nutrients and bioactive molecules but also carrying two major drawbacks. First, the protein components and the presence of xenocarbohydrate contaminants in FBS, which can be internalized by MSCs, may induce immune reactions in the patients and lead to MSC transplantation failure.10,11 Second, some FBS batches may carry the risks of being contaminated by adventitious pathogenic agents such as viruses12 as well as the bovine spongiform encephalopathy prion responsible for variant Creutzfeldt-Jakob disease in humans,13 thereby raising concerns on use for therapeutic applications.14 The use of materials from bovine origin within therapeutic protocols is discouraged by regulatory authorities and in international guidelines.14,15 In addition, the use of FBS is complicated in routine practice by lot-to-lot variability and decreased availability due in part to more stringent regulations or geographical origin. The development of safe culture conditions complying with pathogen safety requirements and good manufacturing practices is one key element to consolidate the success of cellular therapies.16-19 The culture medium itself is one most critical good manufacturing practices factor in MSC cultures. Serum-free medium is unable to promote MSC expansion unless several recombinant human (rHu) growth factors (GFs) such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF)-b, and epidermal growth factor (EGF) are added to the medium.20 However, 1) defining the optimal amount of GF is difficult, 2) only a few GFs are licensed for therapeutic use, 3) rHu-GF are expensive, and 4) isolated GF cannot replace the diverse physiological functions of FBS. Interesting research works have evidenced the possibility to replace FBS by autologous or allogeneic, single-donor or pooled human serum or platelet (PLT) releasates for ex vivo expansion of MSCs from different origins,21-24 under conditions where these cells maintain their ability to differentiate into osteogenic, chondrogenic, or adipogenic lineages.23,25-29 The function of human serum and PLT releasates is thought to be linked, at least in part, to their content in GFs, such as PDGFs (PDGF-AA, -AB, and -BB), transforming growth factor-b (TGF-b1 and TGF-b2), EGF, vascular endothelial growth factor (VEGF), FGF, and insulin-like growth factor (IGF), in addition to other vital nutrients and factors required for cell proliferation.24 The use of human-derived blood materials alleviates the immunologic risks of FBS, but the possibility for transmission of blood-borne viruses

remains when using allogeneic human-derived materials, especially when materials from multiple donors are pooled to provide sufficient volume for therapeutic-scale MSC expansion, and to limit individual donor variability.23,24,26 The development of virally inactivated PLT lysate (PL) to supplement culture medium used for MSC expansion seems to be highly desirable. Recently, we have described a procedure to obtain a standardized GF-rich PL preparation that is virally inactivated by a solvent/ detergent (S/D) treatment.30,31 The current study evaluates the feasibility of using such S/D-treated PL (S/D-PL) to substitute for FBS for isolation, maintenance and ex vivo expansion of adipose derived–MSCs (AT-MSCs).

MATERIALS AND METHODS Preparation of S/D-PL PLT concentrates were collected from volunteer donors, who provided informed consent, using a multiple component system and the standard collection procedure of the manufacturer (MCS+, Haemonetics Corp., Braintree, MA), as described previously.30 PCs were stored under mixing at room temperature and processed by S/D using 1% (vol/ vol) tri-n-butyl phosphate (TnBP, Merck KGaA, Darmstadt, Germany) and 1% (vol/vol) Triton X-45 (SigmaAldrich, St Louis, MO) within 24 to 72 hours of collection, as before30 and as summarized in Fig. 1. The S/D-PL fraction was sterile filtered and stored at 4°C for up to 2 weeks until use. GFs were determined using enzyme-linked immunosorbent assay kits (Quantikine, R&D Systems, Minneapolis, MN), residual TnBP by gas chromatography, and Triton X-45 by high-performance liquid chromatography.30,31 To avoid fibrin gel formation in culture medium, the final S/D-PL product was always supplemented with 15 ng/mL heparin (approx. 2-3 IU/mL).

Isolation of AT-MSCs Adipose tissue was obtained by liposuction from four healthy donors aged 20 to 45 years, who provided their informed consent, and after the institutional review board approval protocol of Taipei Medical University, Taiwan. Processed lipoaspirate (PLA) fraction was separated from adipose tissue using a procedure modified from Zuk and coworkers.32 Briefly, the PLA (15-30 mL) was washed with phosphate-buffered saline (PBS) buffer. Washed PLA was digested in PBS containing 0.2% collagenase (C-9891, Sigma) on a 37°C shaking water bath for 30 minutes. Floating adipocytes were aspirated from pelleted AT-MSCs after centrifugation at 400 ¥ g for 10 minutes. Pellets were resuspended in red blood cell lysis buffer (2.06 g/L Tris base, 7.49 g/L [0.1 mmol/L] NH4Cl, pH 7.2) for 10 minutes at room temperature. After resuspension, the washed AT-MSCs were passed through a 40- to 100-mm cell sieve Volume 51, April 2011

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the cell number at harvest by the plated cell number. At the same time, a growth curve of AT-MSCs from three different lipoaspirates was started at Passage 0. A total of 1000 cells/cm2 were plated in five T25 flasks (Greiner Bioone, Monroe, NC). The fold expansion from seeding on Day 1 was calculated and compared for supplements with either 10% (vol/vol) S/D-PL or 10% FBS.

Fibroblastoid colony-forming unit assays AT-MSC frequency in primary cultures at Passages 2 and 3 was determined for all donors by counting fibroblastoid colony-forming units. After isolation from raw lipoaspirates, the stromal vascular fraction cells were plated in six-well plates (Falcon, Becton Dickinson) at low densities of 2 ¥ 103 per well. The medium was replaced weekly, and cultures were stopped on Day 14. The cell layer was then fixed with methanol and stained with a Giemsa solution (Merck, Darmstadt, Germany). AT-MSC frequency was calculated as the mean number of colonies per 1000 cells seeded.

In vitro differentiation assays

Fig. 1. Preparation process of the S/D-PL. For further details see Materials and Methods.

(Becton Dickinson, San Jose, CA). The washed cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM), a low-glucose (LG) culture medium (31600-034, Gibco, Invitrogen Corp., Carlsbad, CA), containing 10% FBS (SH30460, Hyclone, Thermo-Fischer Scientific, Waltham, MA) or the substitute, S/D-PL (at a protein concentration equivalent to that of 10% FBS), and 1% penicillin-streptomycin-neomycin (P4083, SigmaAldrich). Cells (1 ¥ 106-2 ¥ 106 cells/dish) were added into 10-cm culture dishes (430167, Corning, Taipei, Taiwan) and cultured at 37°C, in an atmosphere of 5% CO2 and humid air. After 5 to 7 days, adherent cells were harvested as AT-MSCs by trypsinization and passaging into a new DMEM-LG culture medium containing the same supplements. The medium was replaced every 3 to 4 days, when the cells reach near 80% confluence.

Proliferation kinetics AT-MSCs of donors were expanded using either S/D-PL or FBS supplements both at 10% (vol/vol) concentration. Cells were counted and passaged at a confluence of 70% to 80%. At each passage, the population doubling rate was determined as previously described.3 Fold expansion rates were also determined for Passages 3 through 8 by dividing 772 TRANSFUSION


Differentiation assays into the chondrogenic, osteogenic, and adipogenic lineages were performed for AT-MSCs of donors and of all culture conditions at Passage 1 and then again at Passage 7 essentially as we have described before.33 The goal was to assess whether typical MSC surface markers shown by flow cytometry analysis were retained throughout long-term culture. To initiate the chondrogenic differentiation, AT-MSCs were seeded with a higher cell density (2 ¥ 105/10 mL) in a DMEMLG medium (Gibco) supplemented with 10% FBS (Hyclone) or 10% S/D-PL, 6.25 mg/mL insulin (SigmaAldrich, St Louis, MO), 10 ng/mL TGF-b1 (R&D Systems), and 50 nmol/L ascorbate-2-phosphate (Sigma-Aldrich). Chondrospheres were observed over time and examined by microscopy and detected by Alcian blue stain. Osteogenic differentiation was confirmed by the increase of alkaline phosphatase expression by histochemical staining (85L-3R, Sigma-Aldrich) following manufacturer’s instructions. The formation of a hydroxyapatite matrix was confirmed by the van Kossa stain. Adipogenic differentiation was induced in 100% confluent AT-MSC cultures with three cycles of alternating induction and maintenance medium. The developing lipid vacuoles were stained with Oil Red. All osteogenic and adipogenic media consisted of bullet kits by Cambrex (East Rutherford, NJ), which included osteogenic and adipogenic basal medium in addition to the supplements.

Flow cytometry analysis Cells were analyzed according to the instructions of the flow cytometer manufacturer (FACSCalibur, Becton


Dickinson, Franklin Lakes, NJ). Briefly, an aliquot of 1 ¥ 105 cells harvested from culture dish was labeled with fluorescein isothiocyanate (FITC) or phycoerythrin-conjugated (PE) monoclonal antibodies in 100 mL phosphate buffer for 15 minutes at room temperature. Cell surface markers tested for mesenchymal and white blood cell antigen lineage were CD31-PE, CD29-FITC (Becton Dickinson), CD73PE, CD90-FITC, human leukocyte antigen (HLA)-ABC-FITC, and HLA-DRFITC (Immunotech-Coulter, Marseille, France). CD14-PE, CD31-PE, CD44FITC, CD73-PE, HLA-1-FITC, and HLA-DR-FITC were again reassessed at Passages 5 to 7. A total of 50,000 to 100,000 labeled cells were acquired and analyzed using a flow cytometer running its accompanying software (FACScan and CellQuest, respectively, Becton Dickinson).

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Fig. 2. Microscopic observation (at 100¥ and 200¥ magnification) showing a typical spindle morphology of AT-MSCs cultured in DMEM-LG supplemented with 10% FBS or 10% S/D-PL.

Human cytokine antibody array The S/D-PL and FBS at equivalent protein concentration (determined by the Biuret assay) were compared to evaluate their cytokine profiles using a semiquantitative human cytokine antibody array that detects 120 cytokines in one experiment (RayBio human cytokine antibody array G series 2000, Tebu-bio GmbH, Berlin, Germany). The array consisting of two cytokine antibody–bounded membranes was treated according to the manufacturer’s instructions and incubated with one- to twofold diluted culture medium for 2 hours. All sample measurements were performed in duplicate. The array exhibits specificity to human cytokines but also cross-reacts with some cytokines from bovine origin.

RESULTS Morphology Figure 2 shows the typical morphology of the AT-MSCs at Passage 3 when cultured in DMEM-LG basal medium supplemented with either 10% FBS or 10% (vol/vol) S/DPL. Cells were grown to a subconfluent layer (70%-80% confluence). They exhibited a similar typical fibroblast-like, spindle morphology. There were no obvious morphologic differences between S/D-PL and FBS at 10% concentration each.

Growth curves The growth curves of three individual MSCs cultures (A, B, and C) in DMEM-LG basal medium supplemented with

either 10% FBS or 10% S/D-PL are in Fig. 3. S/D-PL– cultured MSCs proliferated faster than FBS-cultured MSCs, as shown by the total cell count measured over a period from 25 to 70 days. Therefore, 10% S/D-PL is capable of supporting the long-term MSC culture at least as well as (A) and better (B and C) than FBS.

MSC immunophenotype The expression of cell surface markers was analyzed by flow cytometry at Passage 7 (S/D-PL; n = 3) or Passage 8 (FBS; n = 3). A panel of nine surface markers (CD29, CD31, CD44, CD73, CDw90, CD105, CD166, HLA-1, and HLA-DR) was used to study the impact of the medium supplement on the immunophenotypic characteristics of AT-MSCs. Figure 4 shows typical flow cytometric histograms evidencing that S/D-PL preserved the major MSC surface markers expressed with FBS at least up to Passage 7.

Differentiation assay At Passage 7, AT-MSCs on 10% S/D-PL or 10% FBS were used for the chondrogenic, osteogenic, and adipogenic differentiation. Figure 4 shows the results of the chondrosphere formation assay using Alcian blue stain at 20, 24, and 47 hours, when cultivating the FBS- or S/D-PLMSCs in a supplemented DMEM low-glucose medium designed for chondrogenesis. S/D-PL was found to Volume 51, April 2011

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The analysis of the cytokine profile in S/D-PL and FBS was performed by a semiquantitative human cytokine antibody array able to detect 120 cytokines. Forty-seven cytokines of 120 were not detectable in S/D-PL. A strong signal was observed with S/D-PL (and FBS) for IGFBP-1, interleukin (IL)-1a, IL-1ra, IL-2, MCP-2, dtk, ENA-78, Fas/TNFRSF6, FGF-4, FGF-9, IGF-BP-6, and IL-1 R4/ST2. Twenty-two cytokines, including intercellular adhesion molecule-1 (ICAM-1), were present at higher concentrations in S/D-PL than in FBS while only two cytokines (angiopoietin-2 and b-FGF) were found at lower concentrations (Table 1).

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Fig. 3. Proliferation profile of AT-MSCs from three individual isolates (A, B, and C) cultured over 25 to 70 days in DMEM-LG supplemented with 10% FBS (䊊) or 10% S/D-PL (䊉).

enhance the MSC chondrosphere formation in chondrogenesis culture. Chondrosphere formation was initiated sooner (approx. 12 hr) and completed sooner (20 hr) than for the FBS-MSC culture (24-47 hr). Osteogenic differentiation, as assessed by alkaline phosphate expression and hydroxyapatite formation, and adipogenic differentiation, as judged by lipid vacuole formation, was observed in both culture types, without obvious differences whether MSCs were cultured with S/D-PL or FBS (not shown). 774 TRANSFUSION


MSCs have the potential to play a major role in the field of cell therapy to address numerous diseases although some hurdles remain.34 So far, most cell therapy approaches using MSCs are based on methods that use FBS as a gold standard supplement of cell culture medium. However, the use of a material from bovine source presents the drawbacks for patients of risks of cross-species exposure to protein,10,11 viruses, or prions.14,15 Therefore, substituting the human blood–derived fraction for FBS appears preferable in clinical-scale expansion of MSCs. Recent studies have shown that marrow or AT-MSCs can be efficiently propagated in growth medium supplemented with human PLT releasates, under conditions where they maintain clonogenic characteristics and ability to differentiate toward osteogenic, chondrogenic, adipogenic, or hepatocyte cellular lineages.23,25,35,36 The use of human serum or PL of autologous origin is a safe alternative but may be limited by 1) the substantial volume needed for clinicalscale expansion of MSCs; 2) the patients’ health conditions that may prohibit blood donation; and 3) individual donor variability, which may lead to divergent GF concentrations and inconsistency in MSC expansion. The preparation methods and use of pooled PLs have been described and shown to support excellent MSC proliferation.24,26,37 In such procedure, 15 to 50 single-donor PLTrich plasma donations are pooled and subjected to one or several freeze-thaw cycles to induce the release of the GF. The PL is then centrifuged to remove the PLT fragments. Pooled PL lots are expected to have more consistent quality and are available in higher volume than singledonor products. As the pooling statistically increases the risk of viral contamination, a viral inactivation step, in addition to the standard donors screening and donation testing measures, is highly desirable for routine clinical applications, as is the case for many years for any pooled blood products.38,39 We have therefore decided to examine the possibility of using virally inactivated PL as supplement of animal serum-free medium for stem cell maintenance and expansion. Several viral inactivation


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Fig. 4. Flow cytometric characterization of AT-MSCs. Comparison of the expression of surface proteins of AT-MSCs cultured in FBS at Passage 8 or S/D-PL at Passage 7 analyzed by flow cytometry.

process includes centrifugation and a 0.2-mm filtration step that remove blood cell membrane fragments, therefore BDNF EGF I-309 Leptin LIGHT MIP-1-delta reducing risks of alloimmunization in NAP-2 PDGF-BB RANTES Acrp30 Angiopoietin-2* Axl treated patients. Such preparation b-FGF* EGF-R ICAM-1 IGF-BP-6 IL-6 R MAS-a Sgp130 sTNF RII sTNF-R1 TIMP-1 TIMP-2 TRAIL-R3 contains high concentration of the * Higher concentration in FBS than in S/D-PL; others cytokines were present at higher main PLT GFs (TGF-b1, PDGF, EGF, concentration in S/D-PL than in FBS. VEGF, IGF), plasma proteins,30 and other BDNF = brain-derived neurotrophic factor; ICAM-1 = intercellular adhesion molecule-1. important compounds such as cytokines, hormones, and attachment factors present in plasma or released by the PLTs. While S/D-plasma for transfusion virally inactivated procedures could be considered for PLT preparations for by the 1% TnBP/1% Triton X-100 combination has been MSC expansion, including those such as psoralen/UV or found to have lower a-2-antiplasmin44 and a-1 antitriboflavin/UV treatments, that are applied to therapeutic 40 PLT units for transfusion. However, these technologies rypsin45 functional activity, this detrimental impact seems aim at altering pathogen nucleic acids and the conditions to be associated to the use of Triton X-10045,46 and has not 41 have been developed for preserving PLT integrity, therebeen found when using Triton X-45,47,48 which we used in fore limiting the release of the GF from the a-granules. The our process. As S/D treatment has no impact on nonenreasons why we selected the S/D treatment are directly veloped viruses, in particular hepatitis A virus and parlinked to the fact that it is a well-established procedure to vovirus B19, minipool nucleic acid testing of these two destroy lipid-enveloped viruses that 1) preserves the funcviruses in the starting PLT concentrates, as routinely pertional activity of most proteins,38,40,42 2) induces PLT lysis formed in the plasma fractionation industry and approved by regulatory authorities,42,49 would be recommended. and massive release of GF,31 and 3) can be readily applied to pooled PLT concentrates for small-, medium-, or largeTo our knowledge, no study has ever been done to scale production as it has been for plasma products.43 evaluate the use of virally inactivated human PL for MSC expansion. In this study, we evaluated the S/D-PL prepaRemoval of the S/D agents can be achieved by oil ration obtained as described previously.30 This preparaextraction and hydrophobic interaction chromatography, 43 a mild process already used in the plasma industry. The tion has a total protein content close to 35 g/L and has TABLE 1. List of cytokines present in different quantities in FBS and S/D-PL, as detected by cytokine antibody array


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been used as such without concentration step. Our data suggest that it is feasible to use such S/D-PL to replace FBS for AT-MSC maintenance and expansion, thereby allowing to get MSC for clinical use in the complete absence of animal proteins. When cultured in a medium supplemented with 10% S/D-PL, AT-MSCs exhibited a spindle morphology similar to that obtained when using FBS. We did not observe the smaller and more fusiform shape and mesh-like growth pattern at Passage 3 to 4 noticed in another study that used thrombin-activated PLT-rich plasma.23 Growth curves over seven passages showed that MSCs expand in a medium supplemented with this S/D-PL at least as well as, if not better than, when using FBS supplement, confirming the data obtained by other groups using non–virally inactivated PL to expand stem cells from marrow or adipose tissues.21-23,25-29 S/D-PL– expanded AT-MSCs exhibited a qualitative and quantitative immunophenotype profile at Passage 7 identical to that found for FBS-AT-MSCs cultures, with similar cell expression percentages. The same was found with AT-MSCs cultured with non–virally inactivated thrombinactivated PLT-rich plasma or frozen-thawed PL.23,35 The differentiation potential of S/D-PL-MSC toward the chondrogenic, osteogenic, and adipogenic cell lineages was evaluated at Passage 7 and found to be maintained. Interestingly, the chondrogenic differentiation occurred faster with S/D-PL. To our knowledge, this is the first time such phenomenon is reported using AT-MSCs and PLT-derived materials as supplement. The chondrosphere formation is due to the condensation of precartilagious cells in early chondrogenesis. Cell adhesion molecules are also involved in the precartilaginous mesenchymal condensations on the chondrosphere formation and chondrogenesis. A higher concentration of intercellular adhesion molecule-1 was found in the S/D-PL cytokine profile (Table 1) that may contribute in part to the acceleration of the chondrosphere formation (Fig. 5). It could possibly be related to the difference in the concentration of some cytokines and chemokines in S/D-PL and FBS. The mechanism by which S/D-PL kinetically enhances the MSC chondrosphere formation in the chondrogenesis culture remains to be evaluated. Differentiation potential toward the osteogenic and adipogenic lineages was also preserved for S/D-PL cultures AT-MSCs. The cytokine antibody array study identified a number of similar concentration of cytokines between S/D-PL and FBS. Differences have also been noted, in particular in the concentration of 24 cytokines, that will deserve more in depth studies. In conclusion, this study demonstrates the feasibility of using S/D-PL to maintain and to enhance AT-MSC expansion ex vivo at least as well as FBS. S/D-treated PLT preparation does not compromise the differentiation capacity, does not alter the immunophenotype of MSCs, and accelerates chondrogenesis. Although further experi776 TRANSFUSION


Fig. 5. Representative images of chondrogenic differentiation showing that chondrosphere formation was enhanced and took place faster when AT-MSCs were cultured with 10% S/D-PL (20 hr) compared to 10% FBS (47 hr).

mental studies are mandatory, these data provide clues for the large-scale production of a standardized virally inactivated animal-free growth medium supplement that could facilitate cell therapy protocols. It also provides elements to support the prospect for dose escalation clinical studies aiming at defining the effective MSC dose for different indications.

ACKNOWLEDGMENTS This work was supported in part by the National Science Council of Taiwan (NSC-98-2011-B038-006) and by a collaboration among three laboratories in Taiwan and in France. We thank Wu Yu-Wen and Weng Wang-Jung and for technical support in the preparation and analysis of S/D-PL.

CONFLICT OF INTEREST The authors state that they have no conflict of interest.

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