In regenerative medicine mesenchymal stem cells are gaining importance because they have a lot of possibilities to become various different cell lineages [5, 13]. For their purpose in medicine it is important that it is possible to store them in a stable state for a long time without losing viability and the ability to differentiate. Cryopreservation is a good possibility for that. With this method today it is possible to store stem cells in a stable state for a long time . But there is the need of CPAs for this purpose, which often has the disadvantage that they are toxic for the cells. The most popular CPA is DMSO which is mostly used in concentrations of 5 to 10 % . Because of its toxicity there is a need to find alternatives. In this work an icebindingprotein from an insect was tested as such an alternative.
Stem cells are cells which have the possibility of an asymmetric division, resulting in another stem cell and a differentiated cell . One type of stem cells is mesenchymal stem cells. They were first described by Conheim 130 years ago . These stem cells can differentiate in all cells of the mesenchymal tissue, but not the mesoderm itself as embryonal stem cells can . For this reason they are often referred to as mesenchymal (or multipotent) stroma cells. Mesenchymal tissue is for example adipocytes, chondrocytes and osteoblasts . The mesenchymal stem cells are exvivo multipotent . This experiment was conducted with mesenchymal stem cells of the amnion from the common marmoset monkey Callithrix jacchus . Although they can be stored for a long time, this storage can also influence the differentiation potential or genetic/epigenetic patterns of the cell. For this reason it is necessary to find a way to avoid these damages . Especially the ice crystallization is a big problem during the freezing- and thawing process. Most of the time the extracellular water freezes faster than the intracellular water, because the nucleation in the cell is slower. These effects lead to a dehydration of the cell [2, 8]. To avoid this CPAs are needed. However most of the ones which are used today are toxic for the cells . A possible alternative are the so called icebindingproteins (IBPs). These proteins were discovered a long time ago, firstly in fish which can live at temperatures lower than the freezing point of their blood . Until today they have been found in a lot of different organisms. What all these proteins have in common is a hydrophil surface, an amphipathic structure and a fleet part on the surface for binding the ice . These proteins bind to an icecrystal in a way of a keylock mechanism . Thereby the morphology of the icecrystal changes and it stops growing . This work relates to an IBP which was found in the snowflea Hypogastrura harveyi. This protein has two isoforms of which the bigger one has a molecular weight of 15.7 kDa. It has the possibility to drop the freezing point of water to 6 °C .
2.1 Culturing bacteria and protein production
First of all the IBP of Hypogastrura harveyi was produced heterologous in the model organism Escherichia coli.
E. coli BL21 (with the vector pET21(a)+sfAFP) was cultured in LBmedium with Ampicillin (100 μg/mL) at 37 °C on a shaker in two successive steps until an OD600 of 0.6. After cooling for 1 h to 16 °C, the protein production was induced with IPTG (100 μg/mL). An incubation at 16 °C and 200 rpm over night followed. The bacterial culture was centrifuged at 5000 xg and 4 °C for 10 minutes, the super-natant was discarded and the pellet resuspended in Lysisbuffer (100 mM NaCl, 50 mM TrisHCl, pH 7.5). Digestion was achieved by adding Lysozym for 1 h on ice and subsequent ultrasonics. After centrifugation (4000 xg, 4 °C, 15 minutes) the protein containing supernatant was filled in dialysis tubes and covered with Polyethylenglycol. This process was continued until the volume in the dialysis tubes was about half of the original size. The proteins were filled in a 50 mL flask and stored at 20 °C.
2.2 Mesenchymal stem cells
2.2.1 Stem cell cultivation and cryopreservation
Mesenchymal stem cells from Callithrix jacchus (cjMSCs) were cultured in MSCmedium until 80 % confluence was reached. After detaching with Trypsin/EDTA cells were counted in an automatic cell analyzer ViCell XR. This result was used to calculate the amount of solution to get the requested number of cells for further experiments.
The required amount of 1 000 000 cells was resus-pended in MSCmedium (1600 μL). The cryoprotectant solutions were added double concentrated. All the next steps were done on ice. 1600 μL of the cryoprotectant solutions were filled in a syringe and applied to the cells dropwise. The cryoprotectant solutions were 2.5 % DMSO, 5 % DMSO, 35 % DMSO, the crude extract from the bacteria (Pellet in Lysisbuffer), the purified inclusion bodies of the bacteria, 2.5 % DMSO with crude extract and 2.5 % DMSO with purified inclusion bodies. After resuspension of the mixtures the cells were aliquoted into cryovials (1 mL per vial). They were cryopreserved in a programmable freezer (CM2000, Carburos Metálicos, Spain) with a cooling rate of 7.5 K/min until 30 °C followed by 3 K/min until 80 °C. Afterwards they were transferred into a box and stored at 150 °C .
For thawing the cryovials were put in a 37 °C warm waterbath until only a small icecrystal remained. Then they were put on ice and thawed there completely. The thawed cells were transferred to a 15 mL tube and 5 mL cold MSC-medium was added dropwise. The resuspended cells were centrifuged at 235 xg for 10 min. The supernatant was discarded and the pellet was initially resuspended in 1 mL prewarmed MSCmedium. This cell suspension was then added to 10 mL prewarmed MSCmedium in a 75Tculture flask and spread equally. They were then incubated at 37 °C and 5 % CO2 for 20 h.
2.2.2 Analysis of cryoprotective properties
All tests were done in 24Wellplates with 20000 cells per Well after 20 h incubation at 37 °C and 5 % CO2.
For the trypanbluedye the medium was removed and the cells were washed twice with 400 μL 1x PBS. Then 200 μL Trypsin/EDTA was added, spread equally and incubated at 37 °C and 5 % CO2 for 3 minutes. The detachment process was controlled visually under a microscope. The reaction was stopped with addition of 800 μL MSC-medium. Then the resuspended cells were transferred to a 1.5 mL tube, centrifuged and the supernatant was discarded. The pellet was resuspended in 200 μL MSC-medium. Out of that solution 10 μL were mixed with 10 μL trypanblue. Immediately after mixing about 10 μL of the mixture were filled in a Neubauer cell chamber and the cells were directly counted under the microscope. With this result it was possible to calculate the number of membrane intact cells per mL.
For the Hoechststaining the medium also was removed and the cells were washed twice with 400 μL 1x PBS. Then 200 μL Paraformaldehyde (PFA) was added and incubated at room temperature for 10 minutes. Then the PFA was removed, the cells were washed with 200 μL PBS and 400 μL PBS was added. The plates were stored at 4 °C with aluminium foil wrapped around them. After 24 h the PBS was removed and 200 μL of the Hoechstsolution (1 μL Hoechst in 1000 μL PBS) was added. Then it was incubated at room temperature for approximately 15 minutes in darkness. The dye was eliminated and 400 μL PBS was added. Afterwards photographs were taken under a fluorescence microscope (Axiovert 40 C) and analysed with ImageJ.
To estimate the metabolic activity of the thawed cells an MTTTest was done. 40 μL MTT were added to the cells and incubated at 37 °C and 5 % CO2 for 4 h. Then 40 μL formicacid/isopropyl was added and incubated for 1 h (RT) in darkness. Afterwards 100 μL from each well were pipetted in three parallels into a 96Wellplate. The absorbance was measured at 570 nm in a WellPlateReader.
The efficiency of the icebindingprotein as a cryoprotectant for mesenchymal stem cells was tested with three different methods. With a trypanbluedye the membrane integrity was tested. This method allows to differentiate between cells either with perforated or intact cell membrane. The second method was a staining with Hoechst dye which was then analyzed with a fluorescence microscope. With this method the cell nuclei of attached cells are stained so that it is possible to see how well the recultivation of the cells worked after cryopreservation. The third method was an MTTTest. It allows testing the metabolic activity by measuring the absorbance of the media after the cells have metabolized MTT to Formazan.
3.1 Membrane integrity (Trypanbluestaining)
In Figure 1 the results of the trypanbluestaining after cryopreservation and recovery for 20 h can be seen which summarizes the recovery rate of the cells.
The first bar represents the number of recovered cells cryopreserved with 5 % DMSO, which was described earlier as the optimal protocol for cjMSCs , so it is considered as positive control. The recovery rate for these cells corresponds to 100 %. In the mix with crude extract (2) no cells were found (0 %). A recovery rate of 35 % was found for the mix with purified inclusion bodies (3). Bar number four represents the negative control because cells were cryopreserved without any CPA. Here a few cells were found after cryopreservation. In the mixes with crude extract and 2.5 % DMSO (5) and inclusion bodies and 2.5 % DMSO (6) the recovery rate of cells was higher than the positive control. The last bar (K) represents cells which weren‘t cry-opreserved. In this control the recovery rate of the cells is higher than the positive control.
After a statistical test it can be seen that for the mixes with inclusion bodies (3), negative control (4), 2.5 % DMSO and crude extract (5) and respectively inclusion bodies (6) as well as for the control (K) there is a statistical signifi-cance to the positive control.
3.2 Recultivation eflciency (Hoechstdyetest)
In Figure 2 the results for efficiency of recultivation 20 h after cryopreservation are shown. The numbers of attached cells were estimated after staining with Hoechstdye.
The first bar represents the cell number of the cells which were cryopreserved with 5 % DMSO, the positive control. In the mix with inclusion bodies (3) only a few cells were found. In this case there wereńt enough cells after cryopreservation to seed 20,000 cells. In the mixes with 2.5 % DMSO and crude extract (5) and respectively inclusion bodies (6) a similar amount of cells as in the positive control are found. In the unfrozen control (K) only an amount of cells of 2/3 of the positive control can be seen.
After a statistical test it can be seen that only the control (K) has a statistical significance to the positive control.
3.3 Metabolic activity (MTTtest)
In Figure 3 the results for the metabolic activity after cryopreservation measured with the MTTTest can be seen.
The first bar represents the absorbance value of the cells which were cryopreserved with 5 % DMSO, the positive control. In the mix with inclusion bodies (3) a negative absorbance can be seen. In the mixes with 2.5 % DMSO and crude extract (5) and respectively inclusion bodies (6) a higher absorbance value was found than in the positive control. The unfrozen control (K) shows a negative absorbance.
There was no meaningful statistical test done, because the absorbance values only differed in the third decimal place.
Summarizing the results of the cryopreservation experiments with cjMSCs there seems to be a stimulated proliferation by using the crude extract which may also have a metabolic activating effect to the cells. In cryopreservation the inclusion bodies seem to have a slightly higher importance, because then more cells can survive the freezing and thawing process.
As already mentioned, cryopreservation is only possible with a cryoprotectant. Otherwise the survival rate of the cells is very low. For cjMSCs cryopreservation with 5 % DMSO as cryoprotectant is the established protocol . But DMSO is cell toxic, so an alternative is needed. In this work an icebindingprotein of the snowflea Hypogastrura harveyi was used as an alternative.
As results of the experiments it can be said that this protein is not able to replace DMSO, but it is possible to reduce the DMSO concentration from 5 to 2.5 % in combination with the protein. The membrane integrity test with Trypanbluedye exclusion (Figure 1) leads to the conclusion that every tested mix has a different effect on the structural integrity of the cell. But it can be seen that the experiments with 2.5 % DMSO and the crude extract and respectively the inclusion bodies have an even higher membrane integrity than the cells with 5 % DMSO. Figure 2 leads to the conclusion that with 2.5 % DMSO and the crude extract and respectively the inclusion bodies the same recultivation efficiency as with 5 % DMSO can be achieved. Figure 3 represents the results for the metabolic activity. The absorbance values are very low or even negative. This might be due to a technical difficulty during the experiment or there are some substances in the experimental approach which have an effect on the absorbance measure. For the future it is quite interesting to further investigate IBPs from insects as alternative CPA for cryopreservation, because in the literature so far only cryopreservation experiments with IBPs of fish are found. There are studies which show that an IBP of Choristoneura occidentalis reveals a 10 to 100 times higher effect on ice inhibition compared to the IBPs from fish .
Special thanks are going to Andres Buchbender and Julia Struss for technical assistance.
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About the article
Published Online: 2015-09-12
Published in Print: 2015-09-01
Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.