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. 2018 Aug 20;8(1):12439.
doi: 10.1038/s41598-018-30772-4.

Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs

Sangeetha Kandoi  1 , Praveen Kumar L  2 , Bamadeb Patra  1 , Prasanna Vidyasekar  1 , Divya Sivanesan  1 , Vijayalakshmi S  2 , Rajagopal K  3 , Rama Shanker Verma  4
Affiliations

Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs

Sangeetha Kandoi et al. Sci Rep. .

Abstract

Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of cells in the range of 0.5-2.5 × 106 cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Platelet content and preparation of pooled human platelet lysate (pHPL). (a) Illustration showing the release of platelet granule contents which include cytokines, angiogenic factors, proteases and chemostatic factors on lysis (b) Overview of freeze-thaw mechanism of platelet lysate preparation, pooling and long-term storage.
Figure 2
Figure 2
Umbilical cord tissue processing and MSC yield. (a) A schematic representation for isolation of MSC from hUCT using explant and enzymatic dissociation culture employing 20% FBS and 10% PL supplemented medium. (b) Comparison of percentage of viable cell yields from explant and (c) enzymatically dissociated cultures supplemented with 20% FBS and 10% PL using trypan blue dye exclusion. Results are expressed as mean ± SEM of three processed samples. Paired one tailed student’s t test for explant cultures and enzymatic cultures were P = 0.03 and P = 0.49 respectively. *P < 0.05.
Figure 3
Figure 3
MSC characteristics as per ISCT criteria. (a) Plastic adherent MSC with fibroblast like shape. (b) Tri-lineage differentiation potential as shown by oil red O, alizarin red S and safranin O respectively for adipocytes, osteocytes and chondrocytes.
Figure 4
Figure 4
Immunophenotypic analysis by flow cytometry. Representative image of flow cytometry analysis of UC-MSC at passage 2 probed with conjugated antibodies against MSC and hematopoietic markers CD73, CD90, CD105, and CD34, CD45 respectively.
Figure 5
Figure 5
Cell cycle analysis and growth kinetics. (a) Representative histograms of cell cycle in synchronized MSCs (b) DNA contents according to the cell cycle phase G0/G1 phase, S phase and G2/M phase for PL and FBS supplemented MSC cultures. Data are represented as mean of three independent experiments. Paired one tailed student’s t test for SubG1 phase of enzymatic cultures was P = 0.02. (c) Growth rate of UCT-MSC cultured in PL was significantly higher than those in FBS on day 5 by trypan blue dye exclusion (P = 0.05). *P ≤ 0.05.
Figure 6
Figure 6
Immune suppression assay by mixed lymphocyte reaction. Representative bright-field images of positive control (PBMNC with PHA) showing aggregation of cells indicating proliferation of lymphocytes, negative control (PBMNC without PHA) showing no aggregation stating for the absence of proliferation of lymphocytes and PL expanded MSCs co-cultured with allogeneic PBMNC in the presence of PHA showing considerably less aggregates indicative of immunomodulation.
Figure 7
Figure 7
CFSE-based lymphocyte proliferation assay. (a) Flow cytometry analysis exhibited several histograms representing unstained lymphocytes at 0 h (represented by pink histogram), CFSE stained lymphocytes at 0 h (represented by blue histogram), CFSE dilution peak (represented by orange histogram) and proliferation of stained lymphocytes with respect to CFSE expression level (represented by black histogram). When the lymphocytes proliferate, new peaks moving towards the left of the initial peak with reduced dye intensity is seen. (b) Percentage of proliferating lymphocytes co-cultured with MSCs were normalized with control from the CFSE dilution peak. Statistical comparisons were made within the explant and enzymatic groups respectively over different time points using student’s t test. *P < 0.05, **P < 0.01, ***P < 0.005.
Figure 8
Figure 8
Cytogenetic analysis. Normal Q-banding karyotype obtained from at least 20 proliferating cells of FBS and PL cultured MSC preparations of Passage 4 analyzed revealed a normal 46XY karyotype.
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