Stem cell therapy, as a field of study and a promising clinical treatment, is growing exponentially. Among stem cells, MSCs have distinguished themselves as cells with the greatest applied potential because of their unique combination of characteristics including multipotency, accessibility, culture competency and low immunogenicity, immunomodulatory and tissue repair potential (29-31). One of the most important issues in cell therapy is successful maintenance of cells from bench to bedside. It is imperative to have a standard medium suitable for maintaining these cells for an adequate amount of time until they reach the bedside. This must be done without any significant change in the shape or characteristics of these cells before injection. Therefore it is crucial to determine the shelf-life where all their potentials are maintained (26, 32). To date, the best method for long term maintenance of cells and live tissue samples is cryopreservation. In cell therapy today, if we do not want to freeze the cells, we must treat patients on the day cells are harvested. These cells are kept in HSA supplemented normal saline solution and we have shown the extent of their stability in this study (up to 24 hours). Many times it is not possible to treat the patient on the same day due to unanticipated medical issues, distance or other problems for the patient or physician. Under such conditions, the cells must be frozen, but doing so for a few days is costly and will compromise the quality of the cells. Therefore, finding a way to maintain these cells for a few days will be financially and medically invaluable. Furthermore, studies show that fresh cells (not frozen previously) yield better results in therapeutic applications (33). In her 2013 report, Maria Thompson showed that freeze-thawed cells are functionally impaired, as demonstrated by the therapeutic effects, compared to freshly isolated cells (34). A study using human hepatocytes after cryopreservation observed metabolic defects in these cells. This study reported that the cryopreserved hepatocytes had lost most of their capacity for adherence to surfaces. Adhesion to surface plays a defining role in successfully transfusing these cells (35). In hematopoietic stem cell transplantation, researchers have reported use of fresh cells (not cryopreserved cells) in transplant patients result in faster engraftment, less neutropenic fever, and shorter hospitalization time. Chinnaduria et al (36) found that cryopreserved MSC adhesive capacity to fibronectin was reduced by 40% compared to freshly cultured MSC. Cryopreserved MSC binding defect compared to fresh MSC was even greater when adhering to endothelial cells (up to 80%). This observation is consistent with the 60% reduction in cytoskeletal F-actin observed in thawed cells. They also showed that while thawed MSC were not detectable after injection, freshly isolated human MSCs were detectable up to 24 hours after injection into murine liver.
One of the important aspects of MSCs is their immunomodulatory function. Many researchers have explored this capacity in freeze-thawed MSC versus freshly cultured MSCs. They found thawed MSCs have significantly reduced viability, substantially diminished capacity to inhibit in vitro T cell proliferation and decreased sensitivity to IFN-γ compared to freshly isolated cells. The thawed cells showed a relative unresponsiveness to both pro inflammatory and antiinflammatory cytokine signals (21, 37). Fortunately, once these thawed cells are cultured for 24 hours, they do regain much of their immunomodulatory capacities. Therefore, when cryopreserved cells are thawed, they must be cultured for a certain period of time before they are injected into patients. This finding clearly shows there is a difference between freshly cultured and freeze- thawed MSCs. It is important to consider this matter when treating patients with stem cells.
To address this issue, we sought to find a cell medium that would allow longer viability and functionality for freshly isolated and cultured MSC. Our medium of choice was human AB positive plasma. First, it has a human origin, which resolves the animal to human contamination issues. Second, we used AB positive plasma because it lacks blood type antibodies. Finally, plasma is the best medium for preserving the natural state of cells.
Our results show that MSCs isolated from bone marrow, adipose tissue and Wharton’s jelly preserve their stemness in AB+ plasma medium whether they are kept in the fridge or at room temperature conditions. We believe MSCs isolated from adipose tissue preserve their proliferative ability better than MSC cells isolated from bone marrow and Wharton’s jelly. This greater potency for expansion in culture conditions has been noted in other studies (38). The pattern of expansion for MSCs isolated from Bone marrow and Wharton’s jelly is similar but shows a more pronounced drop in proliferation relative to adipose tissue isolated MSCs over time. AB+ plasma is a suitable candidate for keeping these cells for at least 4 days because it has no adverse effect on their biological characteristics and capabilities. This finding will prove invaluable for therapeutic use of these cells.
MSC cells must first be isolated from their source, cultured, expanded and harvested. If the patient and clinical conditions are favorable and transfusion is possible on the same day cells are harvested, they can be suspended in normal saline solution supplemented with 2.5% HSA and 1 U/ml heparin. As long as this solution is injected to the patient within 18 hours, the cells will retain their viability, biological characteristics and growth potential in the body. Although these cells maintain their growth and differentiation potential in normal saline conditions for 24 hours, considering the decrease in viability to below 80% (especially for cells isolated from bone marrow and Wharton’s jelly), it is more reasonable to perform the transfusion within 18 hours. We also tested a few samples with suspension in 5% HSA. The results were similar to cells suspended in 2.5% HSA (Data not shown).
If it is not possible to inject cells within 24 hours after MSCs are harvested, the cells can be kept in AB+ human plasma for at least 4 days without compromising their biological characteristics and viability.
Our findings show that average viability and proliferative potential of cells in this medium remain above the acceptable range (viability of more than 80% and cell confluency above 80% within 20 days), however, some samples showed a decrease in these characteristics after 4 days. Our studies suggest MSCs isolated from adipose tissue can be kept in plasma for up to 7 days, however further validation is needed. Furthermore, although it is possible to keep these cells in plasma medium under fridge and room temperature conditions, we suggest cells be kept in fridge conditions (5+/- 3) because conditions are better maintained.
Suggestions for Further studies
To further confirm the findings of this study, these MSCs can be evaluated for immunomodulatory capabilities and efficacy using in vitro tests on day 3 and day 5.