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Recovery of Human Mesenchymal Stem Cells Following Dehydration and Rehydration
Cryobiology 43, 182–187 (2001) doi:10.1006/cryo.2001.2361, available online at http://www.academicpress.com on
Recovery of Human Mesenchymal Stem Cells Following Dehydration and Rehydration Stephen L. Gordon,*,1 Stephanie R. Oppenheimer,* Alastair M. Mackay,* Jason Brunnabend,* Iskren Puhlev,† and Fred Levine† *Osiris Therapeutics, Inc., 2001 Aliceanna Street, Baltimore, Maryland 21231-3043, U.S.A.; and †UCSD Cancer Center, La Jolla, California 92093-0912, U.S.A. As cell therapies advance from research laboratories to clinical application, there is the need to transport cells and tissues across long distances while maintaining cell viability and function. Currently cells are successfully stored and shipped under liquid nitrogen vapor. The ability to store these cells in the desiccated state at ambient temperature would provide tremendous economic and practical advantage. Human mesenchymal stem cells (hMSCs) have broad potential uses in tissue engineering and regeneration since they can differentiate along multiple lineages and support hematopoeisis. The current research applied recent technological advances in the dehydration and storage of human fibroblasts to hMSCs. Three conditions were tested: air-dried, air-dried and stored under vacuum (vacuum only), and incubated with 50 mM trehalose ⫹ 3% glycerol and then air-dried and stored under vacuum (vacuum ⫹ trehalose). Plates containing dehydrated hMSCs were shipped from San Diego to Baltimore overnight in separate FedEx cardboard boxes. The hMSCs were rehydrated with 3 ml of hMSC medium and were able to regain their spindle-shaped morphology and adhesive capability. In addition, they maintained high viability and proliferation capacity. Rehydrated and passaged cells continued to express the characteristic hMSC surface antigen panel. Additionally, cells showed constitutive levels of mRNA for a stromal factor and, when exposed to reagents known to induce differentiation, demonstrated upregulation of two tissue-specific messages indicative of differentiation potential for fat and bone. While our preliminary findings are encouraging, we still need to address consistency and duration of storage by considering factors such as cell water content, oxygen concentration, and the presence of free radicals. © 2001 Elsevier Science (USA) Key Words: human mesenchymal stem cells; dehydration; vacuum; trehalose; glycerol.
As cell therapies advance from research laboratories to clinical application, there is the need to transport cells and tissues across long distances while maintaining cell viability and function. Guo et al. (6) previously reported that it was possible to desiccate human fibroblasts reversibly for up to 5 days while maintaining viability. Their method made use of the protective effects of trehalose, a disaccharide associated with organisms capable of withstanding desiccation (8, 9, 12). Trehalose was introduced into the cells by infection with an adenoviral vector expressing the trehalose biosynthetic genes otsA and otsB, followed by air-drying and storage at room temperature. Puhlev et al. (11) have explored the effects on viability of a Received October 30, 2001; accepted November 14, 2001. This work was funded by a contract from the Defense Advanced Research Projects Agency. 1 To whom correspondence should be addressed. Fax: (410) 522-6999. E-mail: [email protected]. 0011-2240/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.
number of factors involved in the dehydration and storage of human fibroblasts. Results of their experiments have shown that incubating cells with trehalose and glycerol followed by air-drying and storage under vacuum is superior to the conditions that were previously reported. Here, we report the use of those improved conditions to a cell type other than fibroblasts, i.e., human mesenchymal stem cells (hMSCs). These cells have potential uses in tissue engineering and regeneration that are much broader than those for fibroblasts since they can differentiate along multiple lineages (10) and support hematopoeisis (3). Currently these cells are successfully stored and shipped under liquid nitrogen vapor. The ability to store these cells in the desiccated state at ambient temperature would provide tremendous economic and practical advantage. In this study, we have successfully shipped dried
182
RECOVERY OF HUMAN MESENCHYMAL STEM CELLS
mesenchymal stem cells from San Diego to Baltimore overnight in a standard FedEx shipping box, with retention of cellular viability and function. In the short term, the study reported here opens the door to a simpler and less expensive means of distributing cells. Further improvements in this technology may lead to prolonged storage of cells and tissues without the need for cryopreservation. MATERIALS AND METHODS
hMSCs previously isolated from a bone marrow aspirate, in vitro expanded, and stored under liquid nitrogen at the end of passage 1 (10) were shipped in a liquid nitrogen shipper from Osiris Therapeutics in Baltimore, Maryland to University of California in San Diego, California. Frozen cells were thawed and grown in Dulbecco’s modified Eagle’s medium–low glucose (GibcoBRL) plus 10% fetal bovine serum (HyClone) (hMSC medium) in six-well plates to approximately 300,000 adherent cells per well (65% confluence-passage 3) (Set A) and 400,000 adherent cells per well (95% confluence-passage 2) (Set B). Set A consisted of two trials and Set B of three trials. Each trial contained three six-well plates, with each plate representing one of three dehydration conditions tested. “Air-dried” cells were grown in hMSC medium and dried by aspirating the medium from each well and then exposing cells to room temperature air for 10 min in a laminar-flow biological safety cabinet. “Vacuum only” cells were treated as above but stored under vacuum, while “vacuum ⫹ trehalose” cells were incubated in hMSC medium plus 50 mM trehalose and 3% glycerol for 24 h prior to dehydration (11). Cells were then airdried and stored under vacuum. The vacuum seal was applied by using a commercial food storage vacuum sealer (MagicVac, Flaem, Italy). This machine is capable of removing approximately 90% of the air, thus reducing the amount of oxygen present. The bags can maintain a vacuum of up to 76 cm mercury and have a permeability to water vapor of 4.07 g/m2/24 h. All plates were stored in black plastic to protect them from light.
183
Each trial was wrapped separately and shipped approximately 1 day after dehydration. For Group A, two trials were processed and shipped simultaneously; for Group B, three trials were processed and shipped simultaneously. All five sets of plates were shipped from San Diego to Baltimore overnight in separate FedEx cardboard boxes. The ambient ground temperatures during transit ranged from ⫺1 to 18°C. Following receipt in Baltimore, plates were unwrapped and cells rehydrated by adding 3 ml of hMSC medium to each well and incubating at 37°C, 5% CO2 in a humidified incubator. Cells were fed 3 to 4 days after rehydration and the culture was expanded for 7 days. Gross microscopic observations were made and documented. Cells exposed to air-drying only were clearly nonviable, and no cell proliferation was detected over the course of 7 days. Further efforts to expand these cells were discontinued. For the vacuum-only and vacuum ⫹ trehalose test conditions, proliferating cells were evident and were trypsinized and assessed for viability by trypan blue exclusion on Day 7. These cells were then replated and expanded for further viability testing. Culture purity also was assessed by flow cytometry. hMSCs are typically negative for CD45 (mature hematopoietic marker) and positive for CD105 (endoglin) and CD166 (ALCAM) (5). Rehydrated hMSCs were assayed for the presence of these antigens using a Becton–Dickinson FACS Calibur, and purity was determined as a percentage of cells in the assayed population that contained these markers. Processed cells also were evaluated for retention of hMSC function. By definition, hMSCs are multipotent, i.e., capable of differentiating into several mesenchymal lineages. Rehydrated cells were grown under differentiation conditions for adipogenic (7) and osteogenic lineages (2). RT-PCR analysis was performed to determine the upregulation of early standard markers of adipogenic and osteoblastic gene expression. In addition, expression of a stromal marker was measured in rehydrated, undifferentiated cells.
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Microscopy of Rehydrated Cells Gross microscopic observations for trials in Set A indicated that air-dried non-vacuum-stored cells, regardless of trial, failed to survive the dehydration and rehydration processes. Figure 1A shows a representative example of the appearance of nonviable cells after rehydration. In contrast, healthy, spindle-shaped hMSCs (Fig. 1B) were evident in culture plates stored under vacuum-only conditions, although cell recovery was inconsistent from trial to trial and from well to well within the same plate. Those wells that contained healthy cells, however, had coverage at the well periphery only. Cells treated with vacuum ⫹ trehalose also were seen to survive around the well perimeters only, and, again, growth was inconsistent from trial to trial. However, survival appeared to be slightly higher by comparison to those dehydrated without trehalose and glycerol. In all cases, considerable debris from nonviable cells was noted in each well. Set B trials, which had higher confluence and lower passage prior to dehydration compared to Set A, showed no sign of live cells in air-dried plates, while cells stored under vacuum-only or treated with trehalose and glycerol prior to dehydration showed adherent, spindle-shaped, mitotic cells. Also, as previously observed, vacuum-only plates contained viable cells only at
the periphery of wells, regardless of trial. Where plates from Set B did differ from those in Set A, however, was in the case of the vacuum ⫹ trehalose-treated cells. Here survival appeared to be higher for cells in Set B than similarly treated cells in Set A. Under this condition, in two of the three Set B trials some wells were seen with cell coverage ranging from 50 to 100% of the well and extending beyond the well perimeters and into the center. Viability and Viable Cell Recovery of Rehydrated Cells Cell survival was also quantitated in terms of viability and cell recovery after 7 days. For vacuum-only-treated cells and vacuum ⫹ trehalosetreated cells, regardless of trial or group, viability of cells recovered at that time point was 87.5% in one case and ⬎90% for all others, as determined by trypan blue exclusion. These values compared favorably with the viability seen for hMSCs that have been cryopreserved. Viability remained consistently high with increasing passage. Viable cell recovery was determined by comparing the number of viable cells present at day 7 for each trial and condition to the number of cells initially dehydrated per trial and condition and was used to demonstrate the proliferation potential of each culture. In Set A, viable cell recovery was 6.2 and 1.8% for the vacuum-only condition, first trial and second trial, respectively. In con-
FIG. 1. (A) Nonviable cells appeared granular and two-dimensional even after rehydration; (B) rehydrated viable cells were spindle-shaped and appeared to be three-dimensional. Both (A) and (B) were from different wells within the same trehalose 1 vacuum-treated six-well plate.
RECOVERY OF HUMAN MESENCHYMAL STEM CELLS
trast, percentages calculated for vacuum ⫹ trehalose-treated cells were 18.9 and 20.8, first and second trial, respectively. In Set B for two trials, the calculated percentages were 15.4 and 14.9 for vacuum-only-treated cells as compared to 28 and 45 for vacuum ⫹ trehalose-treated cells, respectively. For the third trial in this set, however, viable cell recovery was low for both treatments, i.e., 2.5% for the vacuum-only condition and 2.0% for the vacuum ⫹ trehalose condition, which quantitatively substantiates the inconsistency from one trial to another that had been observed microscopically and discussed above. Cell Surface Antigen Expression by Flow Cytometry A total of seven samples, three from vacuumonly-treated cells and four from vacuum ⫹ trehalose-treated cells from the combined experi-
185
mental sets, had sufficient cell numbers after passaging to be evaluated by flow cytometry for the expression of CD45, CD105, and CD166 surface antigens. A representative sample result is shown in Fig. 2. All samples tested were found to be negative for CD45 and positive for both CD105 and CD166. Culture purity, also determined by flow cytometry, ranged from 97.2 to 99.7%. Differentiation and Stromal Potential by RTPCR Recovered cells also were tested for both differentiation and stromal potential by RT-PCR. Adipogenesis and osteogenesis in MSCs exposed to specific induction reagents compared to cells grown in hMSC medium is a positive indicator of differentiation (A. MacKay, personal communication). Also, constitutive pro-
FIG. 2. All samples tested were negative for CD45 and positive for both CD105 and CD166 surface antigens as determined by flow cytometry.
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GORDON ET AL.
duction of a standard stromal marker is a positive indicator of stromal characteristics of nondifferentiated cells (A. MacKay, personal communication). The results for all four samples tested are clearly positive for the adipogenic and osteogenic lineages and for the stromal marker (Table 1). These results indicate that the rehydrated hMSCs were capable of functional performance similar to MSCs that had never been dehydrated. DISCUSSION
Clinical applications for human mesenchymal stem cells are based on stromal support for hematopoietic cells and on multilineage differentiation potential. Cultures expanded in vitro consist of adhesive, spindle-shaped cells, with viabilities ⬎90%. Cells demonstrate a CD45negative and CD105- and CD166-positive surface antigen expression pattern and are capable of differentiating along multiple cell lineages. We have shown that hMSCs that have been stored for at least 2 days at ambient temperature in the absence of free water and then rehydrated were able to regain their spindle-shaped morphology and adhesive capability. In addition, they maintained high viability and proliferation capacity. Rehydrated and passaged cells continued to express the characteristic hMSC surface antigen panel. Additionally, cells showed constitutive levels of mRNA for a stromal marker and, when exposed to reagents known to induce differentiation, demonstrated upregulation of two TABLE 1 RT-PCR Analysis of Induced (Osteogenic and Adipogenic Lineages) and Uninduced hMSCs
Sample Group A trial 1 vacuum-only Group A trial 1 vac ⫹ trehalose Group B trial 1 vac ⫹ trehalose Group B trial 3 vac ⫹ trehalose
Adipogenic Induced
Osteogenic Induced
Stromal Marker
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹
Note. Relative scoring of results (all positive).
tissue-specific messages indicative of osteogenesis and adipogenesis. Survival, however, was observed only when air-dried cells were stored under vacuum, consistent with previous experiments (11). Although not conclusive, it has been suggested that improved survival might have to do with a lowered oxygen concentration and the subsequent lack of free radical formation to which cells stored under vacuum would be exposed. In addition, we also observed an increase in viable recovery in trials where cells were exposed to trehalose and glycerol prior to dehydration. Trehalose has been shown to confer protection to both membranes and proteins in bacteria during freeze-drying (8, 9, 12). In addition, glycerol accumulation has been associated with the desiccated state in some organisms (1) and was found to enhance viability of rehydrated fibroblasts when used in conjunction with trehalose (11), although the exact mechanism by which this protection occurs is not known. Results of these initial experiments are promising, yet the inconsistency in cell survival among plates treated under the same condition, and among wells within the same plate, is an issue that we need to address. Several factors such as confluence of cultures prior to dehydration, cell exposure to varying oxygen levels, and cell water content could have contributed to this phenomenon, either individually or in concert, and need to be investigated further. Set B trehalose ⫹ vacuum trials had a higher number of viable cells, compared to plates desiccated under the same condition in Set A. Although passage number was lower for cells in Set B than it was for Set A, it has been shown historically that hMSCs perform similarly in terms of form and function at least to passage 4 (2). Confluence prior to dehydration, on the other hand, has been reported as playing a role in culture survival (11). Since Set A cultures were allowed to grow to only 65% confluence prior to being air-dried, 30% lower than Set B trials, culture density is likely to be the reason for the improved results seen in the latter set. Variation in oxygen levels present even in vacuum-stored plates also may have
RECOVERY OF HUMAN MESENCHYMAL STEM CELLS
caused inconsistencies in results. The method of vacuum sealing these plates may have reduced the presence of oxygen unevenly from trial to trial. If, as hypothesized by Puhlev et al. (11), oxygen were available to generate reactive oxygen species, cellular damage could have occurred especially with desiccated cells where repair mechanisms would most likely be compromised, resulting in lower viability. Water content is another parameter which could drastically alter results and may have played a role in cell survival being predominantly located at well peripheries, where even slightly higher levels of moisture may have increased cell water content levels. In previous published work on fibroblasts under similar dehydrations procedures (11), the available free water content was assessed by Fourier transform infrared spectroscopy to be zero. It should be noted however, that this evaluation did not account for bound water, which may have been present. Consequently, even though all cells appeared dry to the eye, their water content levels may have varied. Water content evaluations were not performed on cells in our current experiment, but will need to be determined for a better understanding of the effects of this factor on membrane phase transition, survival, and long term cell storage (4). In conclusion, our initial experiments clearly demonstrate that human mesenchymal stem cells have the potential to be dehydrated, allowing them to be shipped coast to coast without special cryoprotective packaging and retaining their growth and function characteristics. At this time, however, we still need to address the issue of consistency in terms of our current dehydration process and to determine the relationship of factors such as water content and the presence of free radicals to cell survival in the desiccated state for prolonged periods of time.
187
REFERENCES 1. Baust, J. G. Protective agents: Regulation of synthesis. Cryobiology 20, 357–364 (1983). 2. Bruder, S. P., Jaiswal, N., and Haynesworth, S. E. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J. Cell. Biochem. 64, 278–294 (1997). 3. Cheng, L., Qasba, P., Vanguri, P., and Thiede, M. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34⫹ hematopoietic progenitor cells. J. Cell. Physiol. 184, 58–69 (2000). 4. Crowe, J. H., Crowe, L. M., and Hoekstra, F. A. Phase transitions and permeability changes in dry membranes during rehydration. J. Bioenergetics Biomembranes 21, 77–91 (1989). 5. Deans, R. J., and Moseley, A. B. Mesenchymal stem cells: Biology and potential clinical uses. Exp. Hematol. 28, 875–884 (2000). 6. Guo, N., Puhlev, I., Brown, D. R., Mansbridge, J., and Levine, F. Trehalose expression confers desiccation tolerance on human cells. Nat. Biotechnol. 18, 168–171 (2000). 7. Jaiswal, R. K., Jaiswal, N., Bruder, S. P., Mbalaviele, G., Marshak, D. R., and Pittenger, M. F. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J. Biol. Chem. 275, 9645–9652 (2000). 8. Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., and Crowe, L. M. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl. Environ. Microbiol. 61, 3592–3597 (1995). 9. Leslie, S. B., Teter, S. A., Crowe, L. M., and Crowe, J. H. Trehalose lowers membrane phase transitions in dry yeast cells. Biochim. Biophys. Acta 1192, 7–13 (1994). 10. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999). 11. Puhlev, I., Guo, N., Brown, D. R., and Levine, F. Desiccation tolerance in human cells. Cryobiology 42, 207–217 (2001). 12. Welsh, D. T., and Herbert, R. A. Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli. FEMS Microbiol. Lett. 174, 57–63 (1999).
Recovery of Human Mesenchymal Stem Cells Following Dehydration and Rehydration Stephen L. Gordon,*,1 Stephanie R. Oppenheimer,* Alastair M. Mackay,* Jason Brunnabend,* Iskren Puhlev,† and Fred Levine† *Osiris Therapeutics, Inc., 2001 Aliceanna Street, Baltimore, Maryland 21231-3043, U.S.A.; and †UCSD Cancer Center, La Jolla, California 92093-0912, U.S.A. As cell therapies advance from research laboratories to clinical application, there is the need to transport cells and tissues across long distances while maintaining cell viability and function. Currently cells are successfully stored and shipped under liquid nitrogen vapor. The ability to store these cells in the desiccated state at ambient temperature would provide tremendous economic and practical advantage. Human mesenchymal stem cells (hMSCs) have broad potential uses in tissue engineering and regeneration since they can differentiate along multiple lineages and support hematopoeisis. The current research applied recent technological advances in the dehydration and storage of human fibroblasts to hMSCs. Three conditions were tested: air-dried, air-dried and stored under vacuum (vacuum only), and incubated with 50 mM trehalose ⫹ 3% glycerol and then air-dried and stored under vacuum (vacuum ⫹ trehalose). Plates containing dehydrated hMSCs were shipped from San Diego to Baltimore overnight in separate FedEx cardboard boxes. The hMSCs were rehydrated with 3 ml of hMSC medium and were able to regain their spindle-shaped morphology and adhesive capability. In addition, they maintained high viability and proliferation capacity. Rehydrated and passaged cells continued to express the characteristic hMSC surface antigen panel. Additionally, cells showed constitutive levels of mRNA for a stromal factor and, when exposed to reagents known to induce differentiation, demonstrated upregulation of two tissue-specific messages indicative of differentiation potential for fat and bone. While our preliminary findings are encouraging, we still need to address consistency and duration of storage by considering factors such as cell water content, oxygen concentration, and the presence of free radicals. © 2001 Elsevier Science (USA) Key Words: human mesenchymal stem cells; dehydration; vacuum; trehalose; glycerol.
As cell therapies advance from research laboratories to clinical application, there is the need to transport cells and tissues across long distances while maintaining cell viability and function. Guo et al. (6) previously reported that it was possible to desiccate human fibroblasts reversibly for up to 5 days while maintaining viability. Their method made use of the protective effects of trehalose, a disaccharide associated with organisms capable of withstanding desiccation (8, 9, 12). Trehalose was introduced into the cells by infection with an adenoviral vector expressing the trehalose biosynthetic genes otsA and otsB, followed by air-drying and storage at room temperature. Puhlev et al. (11) have explored the effects on viability of a Received October 30, 2001; accepted November 14, 2001. This work was funded by a contract from the Defense Advanced Research Projects Agency. 1 To whom correspondence should be addressed. Fax: (410) 522-6999. E-mail: [email protected]. 0011-2240/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.
number of factors involved in the dehydration and storage of human fibroblasts. Results of their experiments have shown that incubating cells with trehalose and glycerol followed by air-drying and storage under vacuum is superior to the conditions that were previously reported. Here, we report the use of those improved conditions to a cell type other than fibroblasts, i.e., human mesenchymal stem cells (hMSCs). These cells have potential uses in tissue engineering and regeneration that are much broader than those for fibroblasts since they can differentiate along multiple lineages (10) and support hematopoeisis (3). Currently these cells are successfully stored and shipped under liquid nitrogen vapor. The ability to store these cells in the desiccated state at ambient temperature would provide tremendous economic and practical advantage. In this study, we have successfully shipped dried
182
RECOVERY OF HUMAN MESENCHYMAL STEM CELLS
mesenchymal stem cells from San Diego to Baltimore overnight in a standard FedEx shipping box, with retention of cellular viability and function. In the short term, the study reported here opens the door to a simpler and less expensive means of distributing cells. Further improvements in this technology may lead to prolonged storage of cells and tissues without the need for cryopreservation. MATERIALS AND METHODS
hMSCs previously isolated from a bone marrow aspirate, in vitro expanded, and stored under liquid nitrogen at the end of passage 1 (10) were shipped in a liquid nitrogen shipper from Osiris Therapeutics in Baltimore, Maryland to University of California in San Diego, California. Frozen cells were thawed and grown in Dulbecco’s modified Eagle’s medium–low glucose (GibcoBRL) plus 10% fetal bovine serum (HyClone) (hMSC medium) in six-well plates to approximately 300,000 adherent cells per well (65% confluence-passage 3) (Set A) and 400,000 adherent cells per well (95% confluence-passage 2) (Set B). Set A consisted of two trials and Set B of three trials. Each trial contained three six-well plates, with each plate representing one of three dehydration conditions tested. “Air-dried” cells were grown in hMSC medium and dried by aspirating the medium from each well and then exposing cells to room temperature air for 10 min in a laminar-flow biological safety cabinet. “Vacuum only” cells were treated as above but stored under vacuum, while “vacuum ⫹ trehalose” cells were incubated in hMSC medium plus 50 mM trehalose and 3% glycerol for 24 h prior to dehydration (11). Cells were then airdried and stored under vacuum. The vacuum seal was applied by using a commercial food storage vacuum sealer (MagicVac, Flaem, Italy). This machine is capable of removing approximately 90% of the air, thus reducing the amount of oxygen present. The bags can maintain a vacuum of up to 76 cm mercury and have a permeability to water vapor of 4.07 g/m2/24 h. All plates were stored in black plastic to protect them from light.
183
Each trial was wrapped separately and shipped approximately 1 day after dehydration. For Group A, two trials were processed and shipped simultaneously; for Group B, three trials were processed and shipped simultaneously. All five sets of plates were shipped from San Diego to Baltimore overnight in separate FedEx cardboard boxes. The ambient ground temperatures during transit ranged from ⫺1 to 18°C. Following receipt in Baltimore, plates were unwrapped and cells rehydrated by adding 3 ml of hMSC medium to each well and incubating at 37°C, 5% CO2 in a humidified incubator. Cells were fed 3 to 4 days after rehydration and the culture was expanded for 7 days. Gross microscopic observations were made and documented. Cells exposed to air-drying only were clearly nonviable, and no cell proliferation was detected over the course of 7 days. Further efforts to expand these cells were discontinued. For the vacuum-only and vacuum ⫹ trehalose test conditions, proliferating cells were evident and were trypsinized and assessed for viability by trypan blue exclusion on Day 7. These cells were then replated and expanded for further viability testing. Culture purity also was assessed by flow cytometry. hMSCs are typically negative for CD45 (mature hematopoietic marker) and positive for CD105 (endoglin) and CD166 (ALCAM) (5). Rehydrated hMSCs were assayed for the presence of these antigens using a Becton–Dickinson FACS Calibur, and purity was determined as a percentage of cells in the assayed population that contained these markers. Processed cells also were evaluated for retention of hMSC function. By definition, hMSCs are multipotent, i.e., capable of differentiating into several mesenchymal lineages. Rehydrated cells were grown under differentiation conditions for adipogenic (7) and osteogenic lineages (2). RT-PCR analysis was performed to determine the upregulation of early standard markers of adipogenic and osteoblastic gene expression. In addition, expression of a stromal marker was measured in rehydrated, undifferentiated cells.
184
GORDON ET AL. RESULTS
Microscopy of Rehydrated Cells Gross microscopic observations for trials in Set A indicated that air-dried non-vacuum-stored cells, regardless of trial, failed to survive the dehydration and rehydration processes. Figure 1A shows a representative example of the appearance of nonviable cells after rehydration. In contrast, healthy, spindle-shaped hMSCs (Fig. 1B) were evident in culture plates stored under vacuum-only conditions, although cell recovery was inconsistent from trial to trial and from well to well within the same plate. Those wells that contained healthy cells, however, had coverage at the well periphery only. Cells treated with vacuum ⫹ trehalose also were seen to survive around the well perimeters only, and, again, growth was inconsistent from trial to trial. However, survival appeared to be slightly higher by comparison to those dehydrated without trehalose and glycerol. In all cases, considerable debris from nonviable cells was noted in each well. Set B trials, which had higher confluence and lower passage prior to dehydration compared to Set A, showed no sign of live cells in air-dried plates, while cells stored under vacuum-only or treated with trehalose and glycerol prior to dehydration showed adherent, spindle-shaped, mitotic cells. Also, as previously observed, vacuum-only plates contained viable cells only at
the periphery of wells, regardless of trial. Where plates from Set B did differ from those in Set A, however, was in the case of the vacuum ⫹ trehalose-treated cells. Here survival appeared to be higher for cells in Set B than similarly treated cells in Set A. Under this condition, in two of the three Set B trials some wells were seen with cell coverage ranging from 50 to 100% of the well and extending beyond the well perimeters and into the center. Viability and Viable Cell Recovery of Rehydrated Cells Cell survival was also quantitated in terms of viability and cell recovery after 7 days. For vacuum-only-treated cells and vacuum ⫹ trehalosetreated cells, regardless of trial or group, viability of cells recovered at that time point was 87.5% in one case and ⬎90% for all others, as determined by trypan blue exclusion. These values compared favorably with the viability seen for hMSCs that have been cryopreserved. Viability remained consistently high with increasing passage. Viable cell recovery was determined by comparing the number of viable cells present at day 7 for each trial and condition to the number of cells initially dehydrated per trial and condition and was used to demonstrate the proliferation potential of each culture. In Set A, viable cell recovery was 6.2 and 1.8% for the vacuum-only condition, first trial and second trial, respectively. In con-
FIG. 1. (A) Nonviable cells appeared granular and two-dimensional even after rehydration; (B) rehydrated viable cells were spindle-shaped and appeared to be three-dimensional. Both (A) and (B) were from different wells within the same trehalose 1 vacuum-treated six-well plate.
RECOVERY OF HUMAN MESENCHYMAL STEM CELLS
trast, percentages calculated for vacuum ⫹ trehalose-treated cells were 18.9 and 20.8, first and second trial, respectively. In Set B for two trials, the calculated percentages were 15.4 and 14.9 for vacuum-only-treated cells as compared to 28 and 45 for vacuum ⫹ trehalose-treated cells, respectively. For the third trial in this set, however, viable cell recovery was low for both treatments, i.e., 2.5% for the vacuum-only condition and 2.0% for the vacuum ⫹ trehalose condition, which quantitatively substantiates the inconsistency from one trial to another that had been observed microscopically and discussed above. Cell Surface Antigen Expression by Flow Cytometry A total of seven samples, three from vacuumonly-treated cells and four from vacuum ⫹ trehalose-treated cells from the combined experi-
185
mental sets, had sufficient cell numbers after passaging to be evaluated by flow cytometry for the expression of CD45, CD105, and CD166 surface antigens. A representative sample result is shown in Fig. 2. All samples tested were found to be negative for CD45 and positive for both CD105 and CD166. Culture purity, also determined by flow cytometry, ranged from 97.2 to 99.7%. Differentiation and Stromal Potential by RTPCR Recovered cells also were tested for both differentiation and stromal potential by RT-PCR. Adipogenesis and osteogenesis in MSCs exposed to specific induction reagents compared to cells grown in hMSC medium is a positive indicator of differentiation (A. MacKay, personal communication). Also, constitutive pro-
FIG. 2. All samples tested were negative for CD45 and positive for both CD105 and CD166 surface antigens as determined by flow cytometry.
186
GORDON ET AL.
duction of a standard stromal marker is a positive indicator of stromal characteristics of nondifferentiated cells (A. MacKay, personal communication). The results for all four samples tested are clearly positive for the adipogenic and osteogenic lineages and for the stromal marker (Table 1). These results indicate that the rehydrated hMSCs were capable of functional performance similar to MSCs that had never been dehydrated. DISCUSSION
Clinical applications for human mesenchymal stem cells are based on stromal support for hematopoietic cells and on multilineage differentiation potential. Cultures expanded in vitro consist of adhesive, spindle-shaped cells, with viabilities ⬎90%. Cells demonstrate a CD45negative and CD105- and CD166-positive surface antigen expression pattern and are capable of differentiating along multiple cell lineages. We have shown that hMSCs that have been stored for at least 2 days at ambient temperature in the absence of free water and then rehydrated were able to regain their spindle-shaped morphology and adhesive capability. In addition, they maintained high viability and proliferation capacity. Rehydrated and passaged cells continued to express the characteristic hMSC surface antigen panel. Additionally, cells showed constitutive levels of mRNA for a stromal marker and, when exposed to reagents known to induce differentiation, demonstrated upregulation of two TABLE 1 RT-PCR Analysis of Induced (Osteogenic and Adipogenic Lineages) and Uninduced hMSCs
Sample Group A trial 1 vacuum-only Group A trial 1 vac ⫹ trehalose Group B trial 1 vac ⫹ trehalose Group B trial 3 vac ⫹ trehalose
Adipogenic Induced
Osteogenic Induced
Stromal Marker
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹
Note. Relative scoring of results (all positive).
tissue-specific messages indicative of osteogenesis and adipogenesis. Survival, however, was observed only when air-dried cells were stored under vacuum, consistent with previous experiments (11). Although not conclusive, it has been suggested that improved survival might have to do with a lowered oxygen concentration and the subsequent lack of free radical formation to which cells stored under vacuum would be exposed. In addition, we also observed an increase in viable recovery in trials where cells were exposed to trehalose and glycerol prior to dehydration. Trehalose has been shown to confer protection to both membranes and proteins in bacteria during freeze-drying (8, 9, 12). In addition, glycerol accumulation has been associated with the desiccated state in some organisms (1) and was found to enhance viability of rehydrated fibroblasts when used in conjunction with trehalose (11), although the exact mechanism by which this protection occurs is not known. Results of these initial experiments are promising, yet the inconsistency in cell survival among plates treated under the same condition, and among wells within the same plate, is an issue that we need to address. Several factors such as confluence of cultures prior to dehydration, cell exposure to varying oxygen levels, and cell water content could have contributed to this phenomenon, either individually or in concert, and need to be investigated further. Set B trehalose ⫹ vacuum trials had a higher number of viable cells, compared to plates desiccated under the same condition in Set A. Although passage number was lower for cells in Set B than it was for Set A, it has been shown historically that hMSCs perform similarly in terms of form and function at least to passage 4 (2). Confluence prior to dehydration, on the other hand, has been reported as playing a role in culture survival (11). Since Set A cultures were allowed to grow to only 65% confluence prior to being air-dried, 30% lower than Set B trials, culture density is likely to be the reason for the improved results seen in the latter set. Variation in oxygen levels present even in vacuum-stored plates also may have
RECOVERY OF HUMAN MESENCHYMAL STEM CELLS
caused inconsistencies in results. The method of vacuum sealing these plates may have reduced the presence of oxygen unevenly from trial to trial. If, as hypothesized by Puhlev et al. (11), oxygen were available to generate reactive oxygen species, cellular damage could have occurred especially with desiccated cells where repair mechanisms would most likely be compromised, resulting in lower viability. Water content is another parameter which could drastically alter results and may have played a role in cell survival being predominantly located at well peripheries, where even slightly higher levels of moisture may have increased cell water content levels. In previous published work on fibroblasts under similar dehydrations procedures (11), the available free water content was assessed by Fourier transform infrared spectroscopy to be zero. It should be noted however, that this evaluation did not account for bound water, which may have been present. Consequently, even though all cells appeared dry to the eye, their water content levels may have varied. Water content evaluations were not performed on cells in our current experiment, but will need to be determined for a better understanding of the effects of this factor on membrane phase transition, survival, and long term cell storage (4). In conclusion, our initial experiments clearly demonstrate that human mesenchymal stem cells have the potential to be dehydrated, allowing them to be shipped coast to coast without special cryoprotective packaging and retaining their growth and function characteristics. At this time, however, we still need to address the issue of consistency in terms of our current dehydration process and to determine the relationship of factors such as water content and the presence of free radicals to cell survival in the desiccated state for prolonged periods of time.
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