Effect of Platelet Rich Plasma on Post Cryopreservation Viability, Morphology and Proliferation of Human Umbilical Cord Stem Cells

1Biomedical Master Program, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia 2Department of Anatomy, Faculty of Medicine, Universitas Trisakti, Jl. Kyai Tapa, Jakarta, Indonesia 3Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia 4Department of Anatomy, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia Department of Histology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia


Introduction
Stem cells are primitive cells that are endowed with self renewal capacity and can differentiate into other types of mature cells and thus is regarded as multipotent (Emil et al., 2005). Umbilical cord is a promising source of stem cells that have been attempted to cure various diseases since 1988. Umbilical cord as stem cell source has various advantages compared to other sources as it is easily collected from delivery waste, does not cause adverse effects to donor, devoid of ethical problems and the major advantage is its naive immune property, which may greatly reduce rejection problems (Goldstein et al., 2007). Therefore, cryopreservation method is indispensable, if the stem cells are intended to be used for autologous or allogeneic cell therapy (Goldstein et al., 2007). However, there is no standardized cryopreservation method, especially for umbilical cord derived stem cells (Berz and Colvin, 2012).
There are various protocols for cryopreservation, which differ in the type and concentration of supplement and in cell concentration. These variables may have effects on post cryopreservation cell viability, morphology and proliferation capacity, when the cells are cultured in vitro. Moreover, some protocols use xeno material as supplement that may cause immune response (Mackensen et al., 2000) and stimulation of hapten formation (Martin et al., 2005). Therefore, finding xenofree supplement that is suitable for human umbilical cord derived stem cells is very important and platelet rich Platelet Rich Plasma (PRP) is a candidate (Pawitan, 2012). Murphy et al. (2012) found that PRP was an alternative supplement for murine cord blood derived mesenchymal stem cell cryopreservation.
However, there was no standardized supplement concentration for cryopreservation medium, though some protocols used 10 or 20%. A study compared fibroblast viability after cryopreservation using various FBS concentrations, i.e., 0, 10, 20, 50 and 80% and found that 50 and 80% FBS caused decreased viability Falanga et al. (2004). Another study compared 40 and 70% FBS and found no significant difference in viability and recomended the use of 40% FBS (Nazarpour et al., 2012).

Materials and Methods
This was an experimental analytic in vitro study, which was done in Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital-Faculty of Medicine Universitas Indonesia, from April through November 2014. This study got ethical aproval from the Ethical Commitee of the Faculty of Medicine, Universitas Indonesia (ethical clearance No.665/UN2.F1/ETIK/2014). Stem cells for this study were isolated using multiple harvest explant method  from an umbilical cord that was obtained from a Caesarean section delivery, after the woman signed the informed consent form. The cells were propagated untill passage 1 and 2 to get enough cells for cryopreservation experiments and upon subculture would become passage 2 and 3.

Comparison of Cryopreservation Protocols
We compared eight protocols with variation in type of supplement (PRP and FBS), supplement concentration (10 and 40%) and cell concentration (100,000 and 500,000 cells/mL). The eight protocols can be seen in Table 1. All protocols contained final concentrations of 100U Penicillin/100 µg Streptomycin/mL (Biosera LM-A4118/100) and 0.25 µg Fungizone/mL (Gibco 15290-018) in αMEM (Gibco 12000-014) as basal medium and 10% DMSO (Sigma D2650). The cells used were passage 1 and 2 cells with two replications each and therefore there were a total of 32 groups.

Cryopreservation Procedure
Cryopreservation was done by putting cell and cryopreservation medium containing cryotubes in-20°C for 24 h and then the cryotubes were transfered to -196°C (in liquid nitrogen tank) for one month. After one month, the cryotubes were transfered to 37°C (in a water bath), to thaw the cells. Post thawed stem cells were checked for their viability and subcultured. Cultures of post thawed cells were compared to those of fresh cells. When the cells were 30% confluent, photographs were taken to observe their morphology and cell size that was represented by cell area was measured (in µm 2 ) using Axiocam measuring program. Further, viability at harvest and Population Doubling Time (PDT) was calculated.

Data collection and Analysis
Data collected were post thawing and after culture cell-viability, cell size and PDT. When the data were suitable for parametric test, the differences in cell viability, size and PDT of post thawed cells between the eight protocols and fresh cells were analyzed using Analyses of Variance (ANOVA) from Statistical Product and Service Solution (SPSS) software version 16. However, when the data were not suitable, Kruskal-Wallist test was used. Data of pre and post thawing were compared by paired t test (for parametric data) or Wilcoxon signed rank test (for non parametric data).

Results
Post thawing cell counts were greatly reduced, especially in 100,000 cell concentration protocols (protocol 1, 3, 5 and 7). Therefore, viability tests were done on available remaining cells in each protocols.

Cell Viability
The medians of pre and post thawing cell-viability were 95.17 and 81.81% respectively and Wilcoxon test showed significant difference with a median difference of 13.36%. However, Kruskal-Wallis test showed no significant difference in post thawing and after culture cell-viability between the eight protocols.

Morphology and Cell Size
Subcuture from fresh and post thawed cells of the eight protocols showed similar cell morphology that was fibroblastic (elongated and spindle shaped).
Median of cell size after culture in passage-2 and passage-3 were 2464.5 µm 2 and 2072.19 µm 2 respectively and Mann-Whitney test showed a significant difference, with a median difference of 392.31 µm 2 . Moreover, Kruskal-Wallis test of cell size after culture between the eight protocols and fresh cells showed no significant difference in passage-3 cells, but there were significant differences in passage-2 cells between the eight protocols with fresh cells. Cell size of passage-2 cells after culture from the eight protocols and fresh cells can be seen in Fig. 1. Differences in cell size and p value between the eight protocols and fresh cells can be seen in Table 2.

Population Doubling Time
The PDT after culture of the eight protocols and fresh cells can be seen in Fig. 2. Kruskal-Wallis test showed significant diffence in PDT after culture of the eight protocols and fresh cells. Differences in PDT and p value between the eight protocols and fresh cells can be seen in Table 3.

Discussion
In this study, there was a significant decrease (13.36%) in the median of pre and post thawing cell viability from 95.17 to 81.81%. Post thawing cell viability in this study was relatively higher compared to the study of Polchow et al. (2012) which found that post thawing viability was not more than 70%. The difference might be due to different source of cryopreserved cell, as Polchow et al. (2012) used human vascular umbilical cord cells. Another study by Ginis et al. (2012) found post thawing cell viability of 72 and 80% using 5 and 10% DMSO containing cryopreservation medium respectively, (Ginis et al., 2012) which was similar to our result.
In this study, there was no significant difference in post thawing and after culture cell-viability between the protocols that used FBS and PRP. This fact suggests that PRP is equivalent in preserving post thawing cell viability to FBS.
In this study, after culture cell morphology for cryopreserved cells was fibroblastic (elongated and spindle shaped), similar to the findings of other studies (Polchow et al., 2012;Baksh et al., 2007;Secco et al., 2008;Xiang et al., 2007). We did not find any clusters of cells with endothelial appearance/cobblestone like, which might be due to the cryopreserved cell source that were from passage-1 and passage-2.
Morphology of cryopreserved cells using PRP and FBS supplement was similar and this result was in line with the findings of other studies (Polchow et al., 2012;Xiang et al., 2007). This fact suggests that PRP is equivalent in preserving post thawing cell morphology to FBS.
A study by Scheers et al. (2013) showed increase in cell size with increasing passage, which was supposed as cell aging and therefore it was suggested that small cells were preferable. However, there was no published study that measured cell size.
Cryopreserved cell size after culture showed that cells from passage-3 were significantly smaller compared to those from passage-2, with a median difference of 392.31 µm. This fact might be due to increasing homogeneity with increasing passage, as was found by two studies on mesenchymal stem cells (Doan et al., 2012;Liem et al., 2014).
Cryopreserved cell size after culture of protocol P40-100 cells were significantly smaller compared to those of P10-500, P40-500, F10-100, F40-100 and F40-500 and this fact suggested that PRP 40% as supplement was better in preserving cryopreserved cell size after culture.
Fresh cells from passage-2 were smaller compared cryopreserved cells of protocol P10-100, P10-500, P40-500, F10-100, F40-100 and F40-500 after culture. This finding was different from Bahadori et al. (2009) findings that found cryopreserved MSC morphology after culture was similar until passage-9 and increase in size happened after passage-10, where the cells became flatten and large that indicated aging (Bahadori et al., 2009). In this study, increase in cryopreserved cell size after culture might be due to the property of some viable cells from passage-2 that loss their ability to attach and proliferate, so that seeding of 5000 cells/cm 2 was in fact far smaller and therefore needed more population doubling to become confluent. Moreover, cryopreserved cell concentration in Bahadori et al. (2009) study was 10 6 cellsl/mL that caused decrease in DMSO concentration at seeding compared to this study.
In this study, increase in proliferation rate was in line with decrease in proliferation doubling time. Proliferation doubling time in protocol P40-500 that was smaller compared to those of F10-100 and F40-500 showed that PRP 40% was better than FBS 40% and cell concentration of 500.000 was better than 100.000 in preserving cryopreserved cell proliferation.
A study found no difference in proliferation rate between cryopreserved and fresh MSCs, (Vasconcelos et al., 2012) while another study found higher proliferation rate in cryopreserved compared to fresh cells that was supposed to be due to cell selection (Ginis et al., 2012). In our study, PDT of fresh cells was significantly smaller compared to cryopreserved cells in protocol F10-100 and F40-500. Higher proliferation rate in fresh cells might be due to cryopreservetion injury that was endured by cryopreserved cells due to DMSO and very low cooling effect compared to fresh cells that were relatively intact (Gao and Critser, 2000).
Lower proliferation rate of cryopreserved cells in this study might be due to our cryopreservation procedure that did not use slow cooling method. In our study, we put the cell and cryopreservation medium containing cryotubes in -20°C for 24 h and then the cryotubes were directly transfered to -196°C, while other studies used slow cooling method (Ginis et al., 2012;Vasconcelos et al., 2012).
Greatly reduced cell number after washing step after thawing may cause problems, if cryopreservation is intended to keep the cells for later use. Moreover, too low cell count might cause bias in viability testing and this fact was the limitation of our study.

Conclusion
Platelet rich plasma can be used as FBS substitute in cryopreservation medium and the use of PRP 40% and higher cell concentration is recommended.

Acknowledgement
We are indebted to all staff of Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital-Faculty of Medicine Universitas Indonesia, PT. Kimia Farma and Cellsafe who has assisted in maintaining the lab facility and sample collection. Finally, we would like to appreciate the donor who donated the umbilical cord tissue.

Funding Information
This study was funded by a grant from the Department of National Education of the Republic of Indonesia (PUSNAS 2014, contract number: 2218/H2.R12/HKP.05.00/2014).

Author's Contributions
Noviyanti Goei: Laboratory activities, data collection and analysis, writing the article in Indonesian, final approval.
Isabella Kurnia Liem: Developing the idea, consultant, revising the article, final approval.
Jeanne Adiwinata Pawitan: Developing the idea, supervising laboratory activities, revising and translating the article, final approval.
Dian Mediana: Laboratory activities, proof reading the article, final approval.

Ethics
This study was approved by the Ethical Commitee of Faculty of Medicine Universitas Indonesia-ethical clearance No.665/UN2.F1/ETIK/ 2014.