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. 2019 Jul 15;8(7):724.
doi: 10.3390/cells8070724.

Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells

Mathew Cowper  1 , Trivia Frazier  1   2   3 , Xiying Wu  2   3 , Lowry Curley  2   4 , Michelle H Ma  3 , Omair A Mohiuddin  1 , Marilyn Dietrich  5 , Michelle McCarthy  1 , Joanna Bukowska  1   6 , Jeffrey M Gimble  7   8   9
Affiliations

Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells

Mathew Cowper et al. Cells. .

Abstract

Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute.

Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining.

Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FBS when utilized in cell culture media with respect to proliferation, immunophenotype, and CFU-F frequency. When added to osteogenic, adipogenic, and chondrogenic differentiation media, both supplements showed appropriate differentiation by staining.

Conclusion: HPLs is an effective substitute for FBS in the culture, expansion and differentiation of human ASCs suitable for pre-clinical studies; however, additional assays and analyses will be necessary to validate HPLs for clinical applications and regulatory approval.

Keywords: adipogenesis; adipose-derived stromal/stem cells; chondrogenesis; colony forming unit-fibroblast; fetal bovine serum; human platelet lysate; mesenchymal stem cell; osteogenesis; regenerative medicine.

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Conflict of interest statement

M.H.M. is an employee of Obatala Sciences. T.F. is a co-owner, co-founder, CEO and President of Obatala Sciences and former employee of LaCell. X.W. is co-owner and co-founder of LaCell and Obatala and serves as Vice President for R & D at LaCell. J.M.G. is co-owner and co-founder of LaCell and Obatala Sciences, serves as Chief Scientific Officer of LaCell. T.F., X.W., and J.M.G. are inventors on patents relevant to this field. The remaining authors have no disclosures to declare.

Figures

Figure 1
Figure 1
Effect of the concentration of HPL (Human Platelet Lysate) supplementation on ASC proliferation compared to 10% FBS. Stromal media containing 0.1%, 0.33%, 0.75%, 1.0% PL, and 10% FBS were tested for impact on ASC proliferation. Data is reported as the mean ± standard error. * Significant difference; p < 0.05.
Figure 2
Figure 2
Histograms of flow cytometry detection of surface antigens. The ASCs were culture expanded in stromal media containing either 10% FBS or 0.75% PL and the expression determined for the following surface antigens by flow cytometry: CD29, CD31, CD34, CD45, CD73, CD90, and CD105, with IgG serving as a negative control. The histograms displayed are all derived from a single individual ASC donor and are representative of n = 3 donors. CD: cluster of differentiation.
Figure 3
Figure 3
Effect of 0.75% HPL supplementation on the colony-forming unit-fibroblast assay compared to 10% FBS per 102 ASC. Stromal media containing 0.75% PL and 10% FBS were tested for impact on ASC colony-forming unit-fibroblast count. Data are reported as the mean ± standard error.; n = 7, one replicate.
Figure 4
Figure 4
Adipogenic, chondrogenic, and osteogenic differentiation ability of ASCs cultured in 0.75% HPL or 10% FBS containing media. (A) Differentiation of ASCs confirmed by staining of two-dimensional cultures with Oil Red O (adipogenesis in the presence of AdipoQual) or Alizarin Red (osteogenesis in the presence of OsteoQual). (B) Differentiation of ASCs confirmed by staining with Alcian Blue (chondrogenesis in the presence of ChondroQual vs. StromaQual controls) and by qRT-PCR analysis of the transcribed total RNA for the mRNAs collagen I (Col I), collagen II (Col II), aggrecan, and matrilin 1 (qRT-PCR reactions were conducted in triplicate).
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