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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.10, No.02, pp 109-113, 2017
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Introduction
Stem cells whether in the form of a crude mixture after isolation, expanded in vitro or differentiated into functional cells have been shown potential to cure various kind of diseases. 1-3 Moreover, mesenchymal stem cells are increasingly used in various clinical trials and have showed promising results. 4-6 However, in vitro over expansion to provide enough number of cells, and cryopreservation to meet the need of ready to use cells might compromise cell quality. A review on the failure of phase III clinical trial, which used over expanded cryopreserved mesenchymal stem cells to cure steroid resistant graft versus host disease, supposed that the failure was due to impaired immunomodulatory functions due to cryopreservation, and senescence due to overexpansion. 7, 8 Therefore, for patients’ safety and prospective comparisons between trial results, cells need to be characterized, and their function and senescent profile need to be analyzed.
Multiple harvest explant method (MHEM) derived umbilical cord mesenchymal stem cells (UC-MSCs) were shown to undergo senescent beginning at passage-10 (P-10), but until P17, the senescent percentage was below 5%. 9 Since for MHEM UCMSCs there are no senescent data after cryopreservation, this study aimed to analyze the senescent profile of cryopreserved MHEM derived UC-MSCs after serial passages. Cumulative population doublings were computed for every passages to provide comparison measures with other previous and future studies.
Materials and Methods
This was an in vitro observational study that was done June April 2014 through January 2015, in Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital - Faculty of Medicine Universitas Indonesia. Ethical approval was obtained from the Ethical Commitee of the Faculty of Medicine, Universitas Indonesia (ethical clearance No. 665/UN2.F1/ETIK/2014).
MHEM derived UC-MSCs were isolated and cultured in platelet lysate (PL) containing medium as described previously,10and were proven to be MSCs by their property that attached to plastics, by their surface markers, and ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. The cells were cryopreserved at passage-1 (P-1) in 10% dimethyl sulfoxide (DMSO) and 10% PL containing alpha minimal essential medium (αMEM). Cell density at cryopreservation was 500,000 cells/mL, and cryopreservation procedure was done at -20o C for 24 hours, and then moved into liquid N2 tank, vapor phase as described previously. 11 After one month, the cells were thawed and subjected to serial passages until P-8, and senescent analysis was done for all passages.
Thawing and serial passages
Thawing was done as quick as possible as described previously. 11 In brief, the cryotubes were immersed in a 37oC water bath. After that, the cells were transferred into a complete medium, washed, and viable and total cells were counted by trypan blue exclusion method. For all passages, the cells were plated in six wells of a 12-well plate with a seeding density of 5000 viable cells/cm2. Upon 80-90% confluent, two wells were harvested and the cells were re-cultured into six wells, and the four remaining wells were subjected to senescent (β galactosidase) staining (Sigma Cat. no CS0030) as described previously.9
Senescent analysis
Five random photographs of all stained wells were taken using an inverted microscope connected to a digital camera, and senescent percentage from each well was recorded. As the total number of cells at 80-90% confluence was difficult to count, only senescent cells were counted, and total number of cells was interpolated using the area of photograph, area of the well, and mean value of total count from the two harvested wells.
Data collection and processing
Data collected were numbers of harvested cells from the two wells from all passages, and numbers of stained cells in the photographs. Observation and data collection of senescent cell number were done in clear and sharp photograph. The data were tabulated according to the passage and well number. The percentages of
senescent cells, and mean and standard deviation (SD) value of the senescent cells in all passages were computed. Further, cumulative population doublings at P-3 through P-8 were computed.
Results
Viability after thawing following cryopreservation was 80%. However, reculture after thawing showed that most cells were floating and only few cells were attached the next day, much fewer compared to passages of fresh cells after harvest. Floating cells were eliminated after medium change. Reculture after thawing took longer time to become confluent compared to passages of fresh cells. Population doubling at P-1, P-2, and P-3 was 4.11, 5.27, and 4.68 respectively
Figure 1. Microphotograph of cells in a P-3 well with non senescent and five senescent cells Senescent (β galactosidase) staining (Sigma Cat. no CS0030), blue color indicate a senescent cell, magnification 100x
No senescent cells were observed at P-2. Senescent cells began to appear at P-3 (Figure 1). Some of the wells that were intended to be stained were over confluent, and were detached after fixation. Further, some photographs were not sharp, so that they could not be analyzed. Therefore, the number of photographs that was analyzed for senescent cells numbers was incomplete. The number of photograph that were analyzed, mean and standard deviation (SD) of percentage of senescent cells, and cumulative population doubling (CPD) from P-3 through P-8 is shown in Table 1.
Table 1. Percentage of senescent cells, number of analyzed photographs, and CPDs in P-3 through P-8
|
P-3 |
P-4 |
P-5 |
P-6 |
P-7 |
P-8 |
Mean |
0.04 |
1.18 |
0.14 |
0.46 |
0.58 |
0.07 |
SD |
0.02 |
1.82 |
0.18 |
NA |
0.91 |
0.07 |
n |
12 |
4 |
18 |
1 |
16 |
15 |
CPD |
14.06 |
16.75 |
21.18 |
26.40 |
29.55 |
34.34 |
P-= passage-, CPD= cumulative population doublings, SD=standard deviation, NA= not applicable, n= number of analyzed photographs
Effect of Cryopreservation and cumulative population doublings on Senescence of Umbilical Cord Mesenchymal Stem Cells
Jeanne Adiwinata Pawitan1,2*, Noviyanti Goei3,4, Isabella Kurnia Liem1,5,
Dian Mediana3,4
1Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital - Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
2Department of Histology, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
3Biomedical Master program, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
4Department of Anatomy, Faculty of Medicine Universitas Trisakti, Jakarta, Indonesia
5Department of Anatomy, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
Abstract : Background: Multiple harvest explant method (MHEM) derived umbilical cord mesenchymal stem cells (UC-MSCs) was shown to undergo senescent beginning at passage-10 (P-10). However, for the same cells, there are no senescent data after cryopreservation and passage. Therefore, this study aimed to analyze the senescent profile of cryopreserved MHEM derived UC-MSCs after serial passages.
Methods: MHEM derived UC-MSCs were isolated and cultured in platelet lysate (PL) containing medium as described previously. The cells were cryopreserved at passage-1 (P-1) in 10% dimethyl sulfoxide (DMSO) and 10% PL containing alpha minimal essential medium (αMEM). Cell density at cryopreservation was 500 000 cells/mL. After one month, the cells were thawed and recultured until P-8 in six 12-well plate. At 80-90% confluent, two wells were harvested and the cells were recultured into six wells, and the four remaining wells were subjected to senescent (β-galactosidase) staining, which was done for all passages. Random photographs were taken from all stained wells, and senescent percentage was recorded.
Results: No senescent cells were observed at P-2. Senescent cells began to appear at P-3. Percentage (mean ± SD) of senescent cells from P-3 through P-8 were 0.04 ± 0.02, 1.18 ± 1.82, 0.14 ± 0.18, 0.49 ± 0.00, 0.58 ±0.91, and 0.07 ± 0.07, respectively.
Results: No senescent cells were observed at P-2. Senescent cells began to appear at P-3. Percentage (mean ± SD) of senescent cells from P-3 through P-8 were 0.04 ± 0.02, 1.18 ± 1.82, 0.14 ± 0.18, 0.49 ± 0.00, 0.58 ±0.91, and 0.07 ± 0.07, respectively.
Conclusion: No senescent cells were observed at P-2 (cumulative population doubling [CPD] > 9.38). Senescent cells began to appear at P-3 (CPD > 14.06), but in all passages until P-8 (CPD >34.34) the senescent percentage was below 5%.
Keywords: MSC, umbilical cord, cryopreservation, passage, senescence.
International Journal of PharmTech Research, Vol.10, No.2, pp 109-113 (2017)
http://dx.doi.org/10.20902/IJPTR.2017.10116