Viability and Apoptosis of Wound Fibroblasts After Cryopreservation

Acknowledgment: This work was supported by the Wound Biotechnology Foundation, Boston, Massachusetts T issue culture for establishing and maintaining cells derived from skin, such as dermal fibroblasts, is a well established technique. The advantage of studying cells from skin in vitro is that it allows investigators to better explore mechanisms of disease and understand how cells respond to certain stimulatory or inhibitory signals. Moreover, cells can be derived from diseased skin and other organs and can be useful in developing insights into abnormalities that may have developed at the cellular and molecular levels. For example, dermal fibroblasts isolated from fibrotic skin have been shown to synthesize increased amounts of collagen in culture over multiple in-vitro passages.¹ Another example is fibroblasts derived from chronic wounds, which have been shown to be senescent and to have low proliferative potential.^2–5 Recently, it has been reported that dermal fibroblasts from chronic wounds have decreased transforming growth factor-b type 2 receptors and faulty levels of phosphorylation of key signaling proteins.³ An additional advantage of being able to culture cells in vitro is that the cells can be cryopreserved in liquid nitrogen for extended periods of time. Thawing of these cultures then allows investigators a way to investigate certain mechanisms of disease without having to obtain additional biopsies from patients or other investigational subjects. Typically, dermal fibroblasts have been cryopreserved in 10- to 20-percent fetal bovine serum (FBS) in Dulbecco’s Modified Eagle’s Medium (DMEM) with an added 10 percent of dimethyl sulfoxide (DMSO),^6,7 although the advantage of a lower concentration of DMSO has been the subject of recent studies.⁸ However, the exact percentage of FBS that is optimal for cryopreservation has not been properly defined. It is unknown whether cryopreservation of cells derived from acute healing wounds would be similar to that observed for cells derived from chronic wounds. In this study, the authors decided to use flow cytometry to determine whether dermal fibroblasts from adult skin display differences in viability and apoptosis depending on the percent of FBS used for cryopreservation. The authors used human dermal fibroblasts derived from acute and chronic wounds from the same patients and developed an assay system that relied on cryopreserving cells for four weeks, allowing them to attach for four hours before flow cytometry. The results of these investigations suggest that high serum concentrations (up to 80 percent) are best for cells obtained from acute wounds, compared to fibroblasts derived from chronic wounds, which appear to do best at 20-percent FBS concentrations. The results also suggest the need for care in deciding on the appropriate FBS concentrations needed for cryopreservation, which may depend on the clinical situation. Methods Fibroblast cultures. Fibroblast cultures were derived from the edge of nonhealing venous ulcers and from acute wounds created on each patient’s ipsilateral thigh skin, as previously described.^3,9 Three patients were used in these studies. Their treatment consisted of a foam dressing and a four-layer compression bandage. The acute wounds on the thigh were first made with a 6-mm punch biopsy of thigh skin, treated with topical mupirocin cream and a nonadherent bandage. They were excised and sutured three days after the initial 6-mm punch biopsy. Cultures were maintained in DMEM plus 10-percent FBS and cryopreserved at an early (three to five) in-vitro passage. Cryopreservation. Cryopreservation was accomplished using 70- to 80-percent confluent cultures and different FBS concentrations of 0, 10, 20, 50, and 80 percent in DMEM plus DMSO (always at 10%). Culture vials were kept in the liquid-air interface of liquid nitrogen. Culture thawing. Fibroblast cultures were thawed and quickly placed in DMEM plus 10-percent FBS. Cells were allowed to attach to tissue culture plastic for four hours. Both attached and unattached cells were harvested for flow cytometry. Flow cytometry. Flow cytometry was accomplished as previously described.¹⁰ Cells were resuspended in propidium iodide (PI) solution at a concentration of 1x10 to the 6 cells per mL. Cells were acquired using CELLQuesc software program with doublet discrimination. At least 25,000 events were acquired for each sample. The cells were then analyzed using the ModFIT LT™ software program (Verity Software House, Inc., Topsham, Maine). Results These studies were undertaken to determine the optimal FBS concentrations for cryopreserving human dermal fibroblasts. In flow cytometry, PI serves as a measure of cell viability, whereas Annexin V helps in determining early apoptosis. Thus, cells that are viable and nonapoptotic would be expected to be negative for propidium chloride and Annexin V. Figure 1 shows that increasing FBS concentrations decrease apoptosis and increase viability in normal fibroblasts remaining unattached after cryopreservation. Thus, there is a dose-dependent effect of serum concentration on the viability and lack of apoptosis of the unattached cells (p=0.0203). It should be noted that all studies were conducted on the cells remaining in the supernatant after four hours. All the cells that attached by that period of time were found to be more than 95 percent viable and less then 5 percent apoptotic (data not shown). Therefore, the studies may represent an assay system for judging the effect of conditions for cryopreservation. Figure 2 shows that the effect on decreased apoptosis is mainly observed at the highest (80%) FBS concentration used (p=0.0317). Representative flow cytometry scatters of unattached normal fibroblasts cryopreserved in 10- or 80-percent FBS are shown in Figure 3. It is clear that in 80-percent FBS a greater percentage of cells are in the left lower quadrant of the diagram, which indicates a lower extent of PI and Annexin V staining. In contrast to what is observed for dermal fibroblasts from acute wounds, fibroblasts cultured from chronic wounds have proven particularly difficult to grow in culture and to be cryopreserved appropriately.^2,3 Therefore, the authors sought to determine whether increasing serum concentrations would also be beneficial in the cryopreservation of chronic wound fibroblasts. For this purpose, the authors obtained dermal fibroblasts from nonhealing venous ulcers from the same patient from whom the acute wound fibroblasts were obtained. In contrast to the results obtained with acute wound fibroblasts, it was found that increasing concentrations of FBS do not result in improved viability after cryopreservation of chronic wound fibroblasts. Figure 4 shows that increasing FBS concentrations, up to 20 percent, decrease apoptosis and increase viability in chronic wound fibroblasts remaining unattached after cryopreservation (p=0.019). However, increasing the serum concentrations used in cryopreservation to 50 or 80 percent actually decreased the extent of fibroblast viability. Discussion Tissue culture and the ability to grow cells from normal or diseased skin remains an important investigational tool. Primary cultures are very useful for determining mechanisms of disease. The ability to cryopreserve these cells obviates the need for repeated biopsy of normal or diseased skin and has resulted in a number of useful studies.^1–4,7 In this investigation, the authors developed a cryopreservation assay system relying on cryopreserved fibroblasts allowed to attach to tissue culture plastic for four hours after four weeks of cryopreservation. Flow cytometry was used to determine the extent of cell viability and early apoptosis in unattached fibroblasts derived from normal acute wounds and from chronic nonhealing wounds in the same patients. With fibroblasts from normal acute wounds, increasing concentrations of FBS used for cryopreservation (up to 80%) led to increased viability and less apoptosis. However, fibroblasts from chronic wounds, which are reported to be senescent,² did not benefit from the higher FBS concentrations, and their viability was best at 20-percent FBS. These results suggest that high FBS concentrations are best for cryopreserving cells derived from acute wounds. However, the appropriate FBS concentrations for certain fibroblast types may need to be determined depending on the disease state and the cell strain involved. There are a number of potential pitfalls of these studies that will need further investigations. For example, this report used four weeks of cryopreservation; it is possible that results may vary depending on the period of time cells remain in liquid nitrogen. Moreover, for the purpose of the cryopreservation assay system, the authors also focused on four hours after the cells were attached. The rationale is that the cells that attached would obviously be considered viable (which indeed was the case) and that sampling of the cells in the supernatant would give a snapshot of cells that had not yet attached and may have either been nonviable or undergoing apoptosis. It is possible that different time periods may need to be used to determine the best assay system for this type of cryopreservation. Another variable that is likely to be important is the cell passage in vitro. Although the authors used a rather uniform in-vitro passage, the extent of cellular senescence, even for acute wound fibroblasts, may be an important variable for optimal cryopreservation. For example, studies that look longitudinally at cellular senescence may be of interest in terms of the cell’s ability to be cryopreserved in liquid nitrogen. Of course, this study focused on FBS concentrations. The contribution of different concentrations of DMSO has already been stated.⁸ Although these potential pitfalls are important, the authors propose that the assay system they developed may be useful in addressing the optimal serum concentrations for acute wound fibroblasts. Also, the authors’ studies indicate that extrapolation of cryopreserving conditions of normal cells to cells obtained from a diseased state may be inappropriate. Thus, chronic wound fibroblasts behave differently in terms of the optimal serum concentrations needed for cryopreservation.