Low DMSO Cryopreservation of Stem Cells Enabled by Macromolecular Cryoprotectants

Mesenchymal stromal (stem) cells have potential in regenerative medicine and modulating the immune system. To deliver any cell-based therapy to the patient, it must be cryopreserved, most commonly in DMSO, which impacts cell function and causes clinical side effects. Here we report the use of a synthetically scalable polyampholyte to rescue the cryopreservation of mesenchymal stromal cells in low [DMSO] cryopreservation. Flow cytometry showed retention of key markers of multipotency comparable to 10% (v/v) DMSO, and the cells could be differentiated, showing this polymer material can be used to improve, or replace, current cryopreservation strategies.


Cryopreservation of cell suspensions
Polymer cryoprotectant was prepared at 2´ final concentration in culture media containing 60 % FBS and 5 % DMSO (final concentration in vial 30 % FBS and 2.5 % DMSO) and allowed to dissolve before sterile-filtering through a 0.2 µm membrane. Cells were removed from culture by treatment with Accutase for 10 minutes at room temperature before neutralisation with complete cell culture media and centrifuged at 180 ´ g for 5 minutes. Supernatant was removed and a sample of cells were diluted 1:1 with 0.4 % trypan blue and the number of viable cells was determined by counting with a haemocytometer. The cell density was adjusted to obtain a cell suspension containing 1 ´ 10 6 cells·mL -1 and a second cell count was performed to obtain an accurate pre-freeze value. 500 µL cell suspension was added to 500 µL cryoprotectant solution in a cryovial and mixed 3 times. Triplicate samples were prepared for each cryopreservation solution; 10 % DMSO, 2.5 % DMSO or 2.5 % DMSO + 20 mg×mL -1 polyampholyte, all containing 30 % FBS. The cryovials were transferred to a CoolCell™ freezing box and frozen at 1 °C·min -1 in a -80 °C freezer. After 2 hours at -80 °C the cryovials were transferred to liquid nitrogen storage for 24 hours. To thaw, cryovials were removed from S4 liquid nitrogen and suspended in a water bath heated to 37 °C. The contents of each vial were added to 9 mL complete media and centrifuged at 180 ´ g for 5 minutes to pellet cells. The supernatant was discarded and the cell pellet was resuspended in 500 µL complete cell media then transferred to individual wells of a 0.1 % gelatin-treated 24-well plate. Plates were maintained in a humidified atmosphere at 37 °C, 5 % CO2 for 24 hours. After 24 hours, total cell recovery and cell viability were assessed by the trypan blue exclusion assay.

Trypan blue exclusion assay
Cells were removed from adherent culture by treatment with Accutase™ for 10 minutes at room temperature then centrifuged at 180 ´ g for 5 minutes. The supernatant was removed and cells were resuspended in 500 µL complete media. A sample of cells was mixed 1:1 in 0.4 % trypan blue and counted using a haemocytometer. Cell viability was calculated as the ratio of unstained cells to the total number of cells post-thaw. Cell recovery was calculated as the ratio of unstained cells to the number of cells initially frozen.

Flow Cytometry Analysis
Cells were frozen in the cryoprotectant solutions as described. Following a 24 hour post-thaw incubation at 37 °C, 5 % CO2, cells were removed from adherent culture by treatment with Accutase™ for 10 minutes at room temperature, then centrifuged at 180 ´ g for 5 minutes.
Supernatant was removed and cells were resuspended in 2 mL staining buffer. An aliquot of cells was removed for counting using the trypan blue exclusion assay. The remaining cells were centrifuged at 180 ´ g for 5 minutes and washed again with 2 mL staining buffer. After

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centrifuging 180 ´ g for 5 minutes, the supernatant was removed and 100 µL staining buffer was added to each sample along with 10 µL of each of the four antibodies with conjugated fluorescent dyes; CD90-APC, CD105-PerCP, CD146-CFS and CD45-PE. Cells were incubated with antibodies for 45 minutes at room temperature before centrifugation at 180 ´ g for 5 minutes. Supernatant was removed and cells were resuspended in 1 mL staining buffer.
Non-frozen, antibody positive (+Ab) controls were prepared using the same procedure. Nonfrozen, antibody negative (-Ab) controls were prepared using the same procedure but substituting 10 µL of each antibody for 10 µL of staining buffer. Isotype controls were prepared by the same method using the corresponding isotype antibodies.

Osteogenic differentiation
Cells were frozen in the cryoprotectant solutions as described. Immediately after thawing, cells were counted and the cell density was adjusted to 1´10 5 cells×mL -1 . 500 µL of each sample was added to wells of a 0.1 % gelatin-treated 24 well plate and incubated at 37 °C, 5 % CO2 for 24 hours (final cell density 5´10 4 cells×well -1 ). Non-frozen controls were prepared at the same density and added to the same plate. After 24 hours, the media was removed and replaced with 300 µL OsteoMAX-XF differentiation media for differentiated wells and 300 µL complete stem cell media containing 8 ng×mL -1 bFGF for undifferentiated controls. Media changes occurred every 2-3 days for 14 days. After 14 days cells were analysed for calcium deposits, indicative of osteoblast cells, by Alizarin Red S staining.

Alizarin Red S Staining
A 2 % Alizarin Red S solution was prepared in deionised water and the pH was adjusted to 4.2 before sterile-filtering through a 0.2 µm syringe filter. Media was removed from all wells and cells were washed with 300 µL dPBS. Cells were fixed with 4 % para-formaldehyde for 15 minutes at room temperature then washed twice with deionised water. 300 µL Alizarin Red S stain was added to each well and incubated in the dark for 15 minutes at room temperature.
The stain was removed and wells were carefully washed four times with deionised water. 500 µL dPBS was added to each well and images were taken using a CKX41 microscope.

Adipogenic differentiation
Cells were frozen in the cryoprotectant solutions as described. Immediately after thawing, cells were counted and the cell density was adjusted to 1´10 5 cells×mL -1 . 500 µL of each sample was added to wells of a 0.1 % gelatin-treated 24 well plate and incubated at 37 °C, 5 % CO2 for 24 hours (final cell density 5´10 4 cells×well -1 ). Non-frozen controls were prepared at the same water. 500 µL dPBS was added to each well and images were taken using a CKX41 microscope.

Low cell density cryopreservation -viability and recovery data
The viability and recovery of cryopreserved hBM-MSCs in different concentrations of DMSO and polyampholyte was initially screened using a cell density of 1´10

Flow cytometry forward and side scatter
The size and granularity of non-frozen control cells and cryopreserved cells was analysed by flow cytometry. Forward and side scatter was recorded for non-frozen, unstained control cells (-Ab), non-frozen, stained control (+Ab) cells and cells frozen with 2.5 % DMSO.

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PERCP, CD146-CFS) and 1 negative marker (CD45-PE). Non-frozen hBM-MSCs, both unstained (-Ab) and stained (+Ab), were provided as controls to observe changes in fluorescence intensity along with isotype antibody controls to determine extent of non-specific antibody binding.

Flow cytometry mean fluorescence values
Mean fluorescence intensity values were extracted from flow cytometry graphs of non-frozen hBM-MSCs and hBM-MSCs cryopreserved with polyampholyte (20 mg×mL -1 ) and/or DMSO Non-frozen MSCs both unstained (-Ab) and stained (+Ab) were provided as controls to observe changes in fluorescence intensity. Results represent three independent repeats (N = 3).
Statistics analysis was completed using an ANOVA test with Tukey post hoc analysis by comparing unfrozen antibody stained hBM-MSCs with all cell freezing conditions.

Light microscope images of thawed cells
Original light microscope images of hBM-MSC frozen with 10 % DMSO, 2.5 % DMSO or 2.5 % DMSO + 20 mg×mL -1 . Cells were frozen at 1 °C×min -1 to -80 °C then transferred to liquid nitrogen for 24 hours. Cells were then thawed at 37 °C in a water bath, transferred to 0.1 % gelatin-treated 24 well plates and placed in an incubator at 37 °C, 5 % CO2 for 24 hours.   Rep S17

Polymer Characterisation
The polymer was analysed by 1 H NMR and 13 C NMR in D2O and reported as follows: Chemical shift (ppm)