- Email: [email protected]
A new material of cryopreserving cell samples
Journal Pre-proof A new material of cryopreserving cell samples Tiantian Liu, Duo Xu, Rong Zhou PII:
S0011-2240(19)30551-6
DOI:
https://doi.org/10.1016/j.cryobiol.2020.02.006
Reference:
YCRYO 4187
To appear in:
Cryobiology
Received Date: 18 November 2019 Revised Date:
14 January 2020
Accepted Date: 13 February 2020
Please cite this article as: T. Liu, D. Xu, R. Zhou, A new material of cryopreserving cell samples, Cryobiology (2020), doi: https://doi.org/10.1016/j.cryobiol.2020.02.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
A New Material of Cryopreserving Cell Samples Tiantian Liu1, Duo Xu2, Rong Zhou2,* 1
School of Public Health, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510310, China
2
State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China * Address correspondence to Dr. Rong Zhou, [email protected]
Summary Cryopreservation was first studied in early twentieth century, and the cryopreservation of cell and tissue samples has become an inseparable process in biology research labs, helping scientists to store living materials and to accumulate specimens. Recently, a new and simplified cryopreservation product, BioFlash Drive™ SP developed by a US firm Fibulas, came to the attention of many researchers. It integrates the sample container with control-released cryoprotectant, and the container itself can achieve slow-freezing in deep freezers. This means no reagents mixing or extra steps for slow-freezing are needed. The design of this product aims to simplify the current cellfreezing procedures and reduce the lengthy and error prone processes. In this research, we compared the post-thaw cell viability using two of the most widely used protocols, with the one with BioFlash Drive™ SP (Fibulas). Cell lines tested in this research include Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™). Results show there is not statistically significant difference in cell viability between the conventional protocols and the new protocols. However, this new protocol is less manual and less time consuming. With this new method, it might be possible for researchers to archive research progress more often, because saving cell can be an easier but still reliable experience.
Key words: Cryopreservation; Vero; HEp-2; viability; Biospecimens; Cell Freezing; Highlights : The present research compares a novel cell cryopreservation kit and conventional cell freezing methods. The results show that there is no significant difference among these methods. The performance of the kit under non-usual extreme conditions is also studied. 1. Introduction Cryopreservation was first studied in the early twentieth century, and has developed and evolved since then[9]. Cryopreserving cell and tissue samples has become an inseparable part in biology research labs, helping scientists to store living materials and to accumulate specimens[12]. On the other hand, in biorepositories and biobanks, cryopreservation plays a crucial role. The value of biological specimens, which cost a fortune to store and keep cold for years, is largely based on how well they can be recovered and revived[2]. Yet the process of preparing, freezing and thawing samples is still a lengthy, tedious and error prone one, often accompanied with unreliable recovery and low viability[5,6,14]. It often includes mixing reagents, preparing freezing containers and cryogenic vials, preprocessing samples, slow-freezing samples, transferring the samples from one freezer to another[5]. Some of these processes involve expensive freezing equipment, such as control-rate freezers[3]. This makes it very inconvenient and sometimes even impossible for scientists and researchers to archive or store progress on biological materials during experiments.
One primary concern in freezing living cells and tissues are intracellular ice and extracellular ice[11,13]. Polge, Smith and Parkes discovered that cells can be damaged by “the mechanical effects”[11]. Ice crystals formed during freezing can pierce or tear apart cells[11]. Lovelock found that “the solution effects” can cause damage to cells, because freezing increases salt concentration in the samples[8]. In order to minimize freezing injury, cooling rate, cryoprotectant, and warming rate are considered key factors to control[4,10]. In some of the widely used cryopreservation procedure, freezing medium is often prepared by mixing 10% DMSO (Sigma-Aldrich) with 90% Fetal Bovine Serum (FBS, Invitrogen)[1]. And then it is used to suspend the cell pellet and is transferred to a cryogenic vial. A freezing container product like Mr. Frosty (Thermo Fisher, USA) or CoolCell (Biocision, USA) is often recommended to keep the cooling rate at -1°C/min in the freezer[1,7]. Yet these procedures take time to gather reagents, consumables and equipment. This can interrupt researchers in experiments and discourage them from archiving intermediate progresses or even saving end products. In our study, we investigated a new tube and a new method for cryopreservation, and validate the performance in both Vero and HEp-2 cells. The result shows that using the new method gives similar results as the conventional method with cryogenic vials (Corning). Yet with this new and less time-consuming method, it is possible to save cells more often, and to archive research progress more frequently. 2. Materials and Methods 2.1. Preparation of Cells Both Vero and HEp-2 were purchased from the ATCC and subsequently maintained in our lab. Vero and HEp-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) with
both 2% 100 mg/mL streptomycin and 10% (v/v) fetal bovine serum (FBS, Invitrogen).
2.2. BioFlash Drive™ Commercial Freezing Kit BioFlash Drive™ SP introduces a new and simplified protocol for cryopreservation (fig 1). It is a cryogenic container with a thermal insulative wall, which, the company claims, can achieve freezing rate at around -1°C/min when it is stored in -80°C freezers or vapor phase nitrogen tanks. Therefore, based on the given Instruction of Use, a control rate freezer is no longer needed. On the other hand, its internal wall is coated with control-released cryoprotectant. As the cell suspension is added to the container, the cryoprotectant, as explained in the product marketing material, will be gradually released and protect the sample in the freezing journey. Therefore, there is no need to mix reagents beforehand.
Our attempts to validate the cooling rate and getting an accurate measurement
of
sample
temperature
using
thermometers
and
thermocouples were not successful, because the sample has a very small volume (1ml) and the thermal dynamics was greatly influenced by the insertion of measurement tools. A more sophisticated system needs to be developed for this purpose, and the complexity of such work is beyond our lab and might deserve more careful effort and maybe another publication. Therefore, we want to limit the scope of this study is to only compare the results of the conventional processes and the new method. Detailed investigations into individual factors that might impact cell viability results, such as cooling rates, warming rates, composition and properties of the cryoprotectant, are left for future efforts.
In this research, we compared the post-thaw cell conditions from two widely used conventional protocols, with those from the BioFlash Drive™ SP protocol. Cell lines tested in this research include Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™).
2.3. Sample Preparation The following sample preparation method is applied to all tested cell lines and in all cryopreservation methods in this research: Prepare the culture medium by mixing DMEM (Invitrogen, Carlsbad, CA) and Fetal Bovine Serum (FBS) (Invitrogen) at a 9:1 ratio. Culture the cell lines with the culture medium above using petri dishes, in 5% CO2 incubators at 37℃. Remove the culture medium.Wash the adherent cells in the petri dishes with PBS (Invitrogen) twice. Dispense 300µl 0.25% trypsin into each petri dish and place the petri dishes in the incubator (5% CO2, 37℃) for 1min.Take out the petri dishes and suspend the cells in each petri dish using 5ml PBS, and transfer the cell suspension to a 15ml centrifuge tube. Centrifuge the centrifuge tube, and remove the supernatant.Resuspend the cells with 6ml PBS, conduct cell viability assay on the sample to obtain total cell count X at this moment.If X is equal to or higher than 4×107, proceed with the next step; if X is lower than 4×107, new cell samples have to be prepared to make sure we have enough cells for this experiment.Transfer 6 ml cell suspension to a centrifuge tube. Label this tube as Tube 1. Transfer 3 ml cell suspension from Tube 1 to another centrifuge tube, labeling it as Tube 2. Centrifuge Tube 1 and Tube 2, and remove the supernatant. 2.4. Freezing for Experiment Group (Tube 1) Resuspend the cell pellet using FBS, which has been precooled to 4℃ beforehand, to give a final cell concentration between 1-2 x 106
cells/ml.Conduct cell viability assay on the sample in Tube 1, record the cell concentration, cell viability, and total cell count.Divide nine Fibulas™ BioFlash Drive™ SP in to three groups (group C, D, E) of three, and label them as C1-C3, D1-D3, E1-E3, respectively. Each group is subject to different test conditions, and the three vials within the same group give a repetition of three times.Transfer 1ml cell suspension from Tube 1 to each BioFlash Drive™ SP vial, and seal the cap.Within 3 minutes after adding samples into the vials, place vials of group C and E into a 5 x 5 cardboard box and place the box into a -80℃ freezer overnight. Subsequently, transfer the cardboard box containing group C and E to liquid nitrogen tank for storage. Samples are not immersed into or in direct contact with liquid nitrogen. (group E will be cultured without washing the cryoprotectant, while group C will be cultured after washing away the cryoprotectant).Place vials of group D into a 5 x 5 cardboard box and place the box in the gas phase section of a liquid nitrogen tank close to the liquid surface. Samples are not immersed into or in direct contact with liquid nitrogen.
2.5. Freezing for Control Group (Conventional methods, Tube 2) Resuspend the cell pellet using 10% v/v DMSO (Sigma-Aldrich) in FBS (Invitrogen) solution, which has been precooled to 4℃ beforehand, to give a final cell concentration between 1-2 x 106 cells/ml.Conduct cell viability assay on the sample in Tube 2, record the cell concentration, cell viability, and total cell count.Divide six Corning 2ml internal threaded cryogenic vials in to two groups (group A, B) of three, and label them as A1-A3 and B1-B3, respectively. The three vials within the same group give a repetition of three times.Transfer 2ml cell suspension from Tube 2 to each cryogenic vial, and seal the cap.Within 3 minutes after adding
samples in to the vials, place vials of group A to a slow-freezing container (Mr. Frosty, Thermo Fisher) prefilled with IPA (Isopropanol, Sigma-Aldrich) according to the product instruction; place vials of group B to another slow-freezing container (CoolCell, Biocision) according to the product instruction.Place both slow-freezing containers to into a 80℃ freezer overnight.Transfer vials of both group A and B to a liquid nitrogen tank for storage. Samples are not immersed into or in direct contact with liquid nitrogen.
2.6. Thawing Method The following thawing method is applied to all tested cell lines and in all cryopreservation methods in this research. Warm the culture medium using 37℃ water bath. After storing in a liquid nitrogen tank for at least 72 hours, take the sample out carefully and warm it by using the water bath. Group A, B,C,and D: Transfer the content of each vial into a centrifuge tube and centrifuge it at 108g for 5 min at 4℃. Discard the supernatant. Suspend the cell with 1ml warm culture medium, and then transfer the cell suspension to one unused well in a 6-well culture plate (Corning™ Costar™ Flat Bottom Cell Culture Plates). Label this well-a (i.e. B2-a). Add 1ml warm culture medium into the well in last step, resuspend the cell evenly in the well. Transfer 1ml cell suspension from the well above to another adjacent hole. Label this well -b (i.e. B2-b). Specifically, for Group E, the washing step is skipped, and the thawed cell suspension is directly transferred to culture wells and diluted with 5ml of culture medium per well Group a. And then transfer 3ml to well Group b.Perform the cell viability assay for Group a after culturing for 3
hours. Repeat the cell viability assay for Group b after culturing for 24 hours.
2.7. Cell Viability Assay The following cell viability assay is applied to all tested cell lines and in both cryopreservation methods compared in this research. In this research, Countess II (Invitrogen) was used for automated cell counting. The machine was in default setting and has been validated by comparison to flow cytometry tests for providing satisfactory cell counting results to our lab when counting Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™), two of the most frequently used cell lines in our lab. We preferred Countess II to manual methods because it provides less countto-count variability. Remove the medium from each culture well to be measured. Dispense 300µl 0.25% trypsin into each culture well and place the culture plates in the incubator (5% CO2, 37℃) for 1min. Take out the culture plates and suspend the cells in each culture well using 0.5ml PBS. For each culture well, mix 10ul 0.4% Trypan Blue with 10ul cell suspension from the culture well and add the sample into a piece of a commercial cell viability slide (Countess II). Insert the slide into a Countess II automated cell counter (Thermo Fisher). Record the cell count number as well as viability, calculate and normalize cell counts in different sample vials: Recovery %=(total cells in a culture well /derived total cells in the corresponding culture well before cryopreservation*) × 100% Viability %=alive cells in a culture well/total cells in a culture well × 100% Cell proliferation %=alive cells (24hr after thawing) in a Group b well/alive cells (3hr after thawing) in a Group a well × 100%
*Because the cells from one vial were divided into two culture wells, the total cells of one vial (before cryopreservation) was divided by 2 to represent the initial total cells in each culture well.
2.8. Statistical analysis Statistical analysis of data was performed using Prism 7 (Graphpad). Wilcoxon signed-rank test was performed because the distribution of the data was unknown, and also because the sample size was relatively small (n=3). P values of less than 0.05 were considered statistically significant.
3. Results 3.1. Experiment group and control groups have comparable performance, after being placed in -80
environment and then
transferred to -150 . Results of group A, B, and C show the performance of two conventional methods and the new method. All samples were placed into -80 prior to -150
freezer
storage, as shown in Fig.2. Fig.2a shows that there is not
significant difference among A, B, and C in terms of cell recovery % 24 hours after thawing. The high value of cell viability % of all groups in Fig.2b indicates that there is no obvious damage to the cell samples. Fig.2c shows cells in Group C has a much higher proliferation % than the control Groups, but no significant difference. 3.2. Both using the new protocol with BioFlash Drive™ SP, placing samples directly in -150 has comparable performance to placing samples in -80
environment and then transferring into -150
environment. As shown in Fig.3a, cells in group C and D do not have significant difference in terms of recovery %. Also, in Fig.3b, the high viability % in
both groups indicates that there is no obvious damage to the cell samples. In Fig.3c, the proliferation % of group C and D do not have significant difference for in each cell type. 3.3. The presence of cryoprotectant preloaded in BioFlash Drive™ SP in cell culturing has different impacts on different cell types. The experiment group E was tested to evaluate the impact of the cryoprotective substances remaining the cell suspension in BioFlash Drive™ SP after thawing. As shown in Fig.4a, cells in group C and E do not have significant difference in terms of recovery %. Also, in Fig.4b, the high viability % in both groups indicates that there is no obvious damage to the cell samples. In addition, as shown in Fig. 4c, Hep-2 cells in group E has a much lower proliferation than cells in group C, indicating different cell types might have different preferences to the cryoprotectant. 3.4. Different methods take different amount of time and steps to complete the cryopreservation process. Based on the methods used in this experiment, we compare the time and steps between the conventional freezing processes and the one with BioFlash Drive™ SP (as shown in the table 1). BioFlash Drive™ SP integrates the cryoprotectant and slow-freezing function into the vial itself. By eliminating the steps of mixing cryoprotectant, transferring samples in and out of slow-freezers, the overall time span for cryopreservation is compressed from across 2 days to about 60 seconds. Although it does not seem that this new method fundamentally changes the principle of slow-freezing, it greatly reduces the time and steps needed to complete the process, making it more convenient to store and archive samples in different research stages. 4. Discussion
According to the results above, there are no statistically significant differences between the conventional protocols and the simplified protocol using BioFlash Drive™ SP in cryopreserving HEp-2 and Vero (Fig.2.). While both using BioFlash Drive™ SP , there is no significant difference of recovery % and viability % between samples first placed in -80
environment and then transferred to -150 , and those directly
placed in -150
environment (Fig.3). This observation should be further
investigated and verified by experimenting with more type of cells. Also, the presence of cryoprotectant from BioFlash Drive™ SP in post-thaw culturing is shown to have different impacts on different cell types. This is a topic worth further investigation.
BioFlash Drive™ SP introduces a new and simplified protocol for cryopreservation(Table 1). With this integrated solution vial, users are able to cryopreserve samples without slow-freezing containers, controlled-rate freezers, or pre-mixing of freezing medium. The results show decent performance on Vero and HEp-2 cells. Although BioFlash Drive™ SP does not show significant difference in results compared to conventional methods, it takes less time, and requires less steps, which make it more convenient for researchers to archive cell samples during experiment process. It may add value to researchers who want to save their samples often.
As mentioned before, due to constrains in time and resources, we were not able to go into details and decompose how BioFlash Drive™ SP achieve similar results as the conventional methods. Although it is claimed to have a thermal insulative layer, which helps it to achieve a 1 /min of cooling curve, it is not verified within this study. Also, the product also claims to have control-release for its cryoprotectant, yet
limited by the scope of this study, we have not studied the concentration change of cryoprotectant over time. We think these are all interesting questions to look into in the future.
5. Conclusion There are no statistically significant differences between the conventional protocols and the simplified protocol using BioFlash Drive™ SP in cryopreserving HEp-2 and Vero. Moreover, using BioFlash Drive™ SP might generate some extra benefits if users choose to place the samples directly into -150 , because this shortcut will not be possible when using conventional methods, which usually require samples to stay in -80 for some time first. Yet the results show that BioFlash Drive™ SP's cryoprotective substances will have different impacts on different cell types. Therefore, users might need to use discretion when using this new product.
This research is important because, to our knowledge, there is no existing study on this new and integrated design for cryopreservation. This research
studied
the
efficiency
and
performance
of
different
cryopreservation methods, both conventional and new, helping researchers to gain insights into different practices, so that they can better choose protocols suitable for their needs.
Acknowledgements: This study was supported by grants from National Key Research and Development Program of China (2018YFC1200100), Entrepreneurship Leadership Project in Guangzhou Development Zone of China(CY2018003), the National Natural Science Foundation of China (NSFC 31570163), the Youth Project of State Key Laboratory of Respiratory
Disease (SKLRD-QN-201713), Guangdong Medical Science and Technology Research Center Project (A2019460) and Scientific Research Project of Guangdong Province Traditional Chinese Medicine Bureau (20201191)
Author Contributions: T.Liu and R.Zhou：designed the experiments and wrote the paper. D.Xu: conducted the experiments. All experiments were performed at State Key Laboratory of Respiratory Disease in Guangzhou Medical University, China. Fibulas did not participate in any experiments.
Conflicts of interest The authors declare that they have no conflicts of interest.
References: [1] ATCC Animal Cell Culture Guide. https://www.atcc.org/~/media/PDFs/Culture Guides/AnimCellCulture_Guide.ashx. (2014). [2] J.M. Baust, W.L. Corwin, R. VanBuskirk, and J.G. Baust, Biobanking: The Future of Cell Preservation Strategies. Adv Exp Med Biol 864 (2015) 37-53. [3] J.G. Day, and M.R. McLellan, Cryopreservation and Freeze-Drying Protocols Volume 38 || Cryopreservation of Algae. 10.1385/0896032965 81-90. [4] J. Farrant, H. Lee, and C.A. Walter, Effects of Interactions Between Cooling and Rewarming Conditions on Survival of Cells. Ciba Found Symp 52 (1977) 4967. [5] R.J. Hay, Preservation of cell-culture stocks in liquid nitrogen. Tca Manual 4 (1978) 787-790. [6] R.J. Hay, Caputo, J., & Macy, M. L. , Preservation of cell cultures in liquid nitrogen. ATCC Quality Control Methods for Cell Lines. (1992). [7] R. Li, R. Johnson, G. Yu, D.H. McKenna, and A. Hubel, Preservation of cellbased immunotherapies for clinical trials. Cytotherapy 21 (2019) 943-957. [8] J.E. Lovelock, The haemolysis of human red blood-cells by freezing and thawing. Biochim Biophys Acta 10 (1953) 414-426. [9] B.J. Luyet, and P.M. Gehenio, Life And Death At Low Temperatures. 3 (1940) 33-99. [10] Mazur, and P., Kinetics of Water Loss from Cells at Subzero Temperatures and the Likelihood of Intracellular Freezing. Journal of General Physiology 47 347-369.
[11] C.A.V. Polge, A.U. Smith, and A.S. Parkes, Revival of Spermatozoa After Vitrification on and Dehydration at Low Temperatures. Nature 164 (1949) 666. [12] M.J. Ryan, and D. Smith, Cryopreservation and freeze-drying of fungi employing centrifugal and shelf freeze-drying. Methods Mol Biol 368 (2007) 127-40. [13] M.J. Taylor, and D.E. Pegg, The effect of ice formation on the function of smooth muscle tissue stored at -21 or -60 °C. 20 0-40. [14] G. Vunjaknovakovic, and R.I. Freshney, Culture of Animal Cells - A Manual of Basic Technique and Specialized Applications, 7th Edition, 2016.
Fig .1. A individual unit of BioFlash Drive™ SP. Fig.2. Post-thaw results of group A, B, and C: 2a, Recovery % 24hours after thawing; 2b, Viability % 24hours after thawing; 2c, Cell Proliferation %. Sample Label: A: IPA box B: foam box C: meta sp-80. Note:Group A and B are control groups of conventional methods; group A uses IPA slow-freezing box while group B uses foam slow-freezing box. Group C and D are experiment groups of the kit, where group C is placed in -80 , group D in -150 . Fig.3. Post-thaw results of group C, and D: 3a, Recovery % 24hours after thawing; 3b, Viability % 24hours after thawing; 3c, Cell Proliferation %. Sample Label: C: meta sp-80 D: meta sp-150 Note: Group C and E are experiment groups of the kit, where group C is placed in -80 . Group E is the only group that is placed in -80
while
the sample within is cultured with the cryoprotectant after thawing. Fig.4. Post-thaw results of group C and E: 4a, Recovery % 24hours after thawing; 4b, Viability % 24hours after thawing; 4c, Cell Proliferation %. Sample Label: C: meta sp-80
E: meta sp-80 cpa
Table 1 Comparison of Two Cell Freezing Protocols Step
Conventional Freezing Process
BioFlash Drive™ SP
1
Mix cryoprotectant
----------
2
Transfer cell suspension to vials
Transfer cell suspension to BioFlash Drive™
3
Prepare a slow-freezing container ---------and slowly freeze samples in -80 . Keep samples there overnight.
4
Take
samples
out
from
slow- ----------
freezing containers 5
Total
Transfer samples to freezers or Put samples directly to -80 freezers or liquid nitrogen tanks
vapor phase nitrogen
Across 2 days
About 60 seconds
Highlights and eTOC Blurb Highlights : The present research compares a novel cell cryopreservation kit and conventional cell freezing methods. The results show that there is no significant difference among these methods. The performance of the kit under non-usual extreme conditions is also studied.
S0011-2240(19)30551-6
DOI:
https://doi.org/10.1016/j.cryobiol.2020.02.006
Reference:
YCRYO 4187
To appear in:
Cryobiology
Received Date: 18 November 2019 Revised Date:
14 January 2020
Accepted Date: 13 February 2020
Please cite this article as: T. Liu, D. Xu, R. Zhou, A new material of cryopreserving cell samples, Cryobiology (2020), doi: https://doi.org/10.1016/j.cryobiol.2020.02.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
A New Material of Cryopreserving Cell Samples Tiantian Liu1, Duo Xu2, Rong Zhou2,* 1
School of Public Health, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510310, China
2
State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China * Address correspondence to Dr. Rong Zhou, [email protected]
Summary Cryopreservation was first studied in early twentieth century, and the cryopreservation of cell and tissue samples has become an inseparable process in biology research labs, helping scientists to store living materials and to accumulate specimens. Recently, a new and simplified cryopreservation product, BioFlash Drive™ SP developed by a US firm Fibulas, came to the attention of many researchers. It integrates the sample container with control-released cryoprotectant, and the container itself can achieve slow-freezing in deep freezers. This means no reagents mixing or extra steps for slow-freezing are needed. The design of this product aims to simplify the current cellfreezing procedures and reduce the lengthy and error prone processes. In this research, we compared the post-thaw cell viability using two of the most widely used protocols, with the one with BioFlash Drive™ SP (Fibulas). Cell lines tested in this research include Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™). Results show there is not statistically significant difference in cell viability between the conventional protocols and the new protocols. However, this new protocol is less manual and less time consuming. With this new method, it might be possible for researchers to archive research progress more often, because saving cell can be an easier but still reliable experience.
Key words: Cryopreservation; Vero; HEp-2; viability; Biospecimens; Cell Freezing; Highlights : The present research compares a novel cell cryopreservation kit and conventional cell freezing methods. The results show that there is no significant difference among these methods. The performance of the kit under non-usual extreme conditions is also studied. 1. Introduction Cryopreservation was first studied in the early twentieth century, and has developed and evolved since then[9]. Cryopreserving cell and tissue samples has become an inseparable part in biology research labs, helping scientists to store living materials and to accumulate specimens[12]. On the other hand, in biorepositories and biobanks, cryopreservation plays a crucial role. The value of biological specimens, which cost a fortune to store and keep cold for years, is largely based on how well they can be recovered and revived[2]. Yet the process of preparing, freezing and thawing samples is still a lengthy, tedious and error prone one, often accompanied with unreliable recovery and low viability[5,6,14]. It often includes mixing reagents, preparing freezing containers and cryogenic vials, preprocessing samples, slow-freezing samples, transferring the samples from one freezer to another[5]. Some of these processes involve expensive freezing equipment, such as control-rate freezers[3]. This makes it very inconvenient and sometimes even impossible for scientists and researchers to archive or store progress on biological materials during experiments.
One primary concern in freezing living cells and tissues are intracellular ice and extracellular ice[11,13]. Polge, Smith and Parkes discovered that cells can be damaged by “the mechanical effects”[11]. Ice crystals formed during freezing can pierce or tear apart cells[11]. Lovelock found that “the solution effects” can cause damage to cells, because freezing increases salt concentration in the samples[8]. In order to minimize freezing injury, cooling rate, cryoprotectant, and warming rate are considered key factors to control[4,10]. In some of the widely used cryopreservation procedure, freezing medium is often prepared by mixing 10% DMSO (Sigma-Aldrich) with 90% Fetal Bovine Serum (FBS, Invitrogen)[1]. And then it is used to suspend the cell pellet and is transferred to a cryogenic vial. A freezing container product like Mr. Frosty (Thermo Fisher, USA) or CoolCell (Biocision, USA) is often recommended to keep the cooling rate at -1°C/min in the freezer[1,7]. Yet these procedures take time to gather reagents, consumables and equipment. This can interrupt researchers in experiments and discourage them from archiving intermediate progresses or even saving end products. In our study, we investigated a new tube and a new method for cryopreservation, and validate the performance in both Vero and HEp-2 cells. The result shows that using the new method gives similar results as the conventional method with cryogenic vials (Corning). Yet with this new and less time-consuming method, it is possible to save cells more often, and to archive research progress more frequently. 2. Materials and Methods 2.1. Preparation of Cells Both Vero and HEp-2 were purchased from the ATCC and subsequently maintained in our lab. Vero and HEp-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) with
both 2% 100 mg/mL streptomycin and 10% (v/v) fetal bovine serum (FBS, Invitrogen).
2.2. BioFlash Drive™ Commercial Freezing Kit BioFlash Drive™ SP introduces a new and simplified protocol for cryopreservation (fig 1). It is a cryogenic container with a thermal insulative wall, which, the company claims, can achieve freezing rate at around -1°C/min when it is stored in -80°C freezers or vapor phase nitrogen tanks. Therefore, based on the given Instruction of Use, a control rate freezer is no longer needed. On the other hand, its internal wall is coated with control-released cryoprotectant. As the cell suspension is added to the container, the cryoprotectant, as explained in the product marketing material, will be gradually released and protect the sample in the freezing journey. Therefore, there is no need to mix reagents beforehand.
Our attempts to validate the cooling rate and getting an accurate measurement
of
sample
temperature
using
thermometers
and
thermocouples were not successful, because the sample has a very small volume (1ml) and the thermal dynamics was greatly influenced by the insertion of measurement tools. A more sophisticated system needs to be developed for this purpose, and the complexity of such work is beyond our lab and might deserve more careful effort and maybe another publication. Therefore, we want to limit the scope of this study is to only compare the results of the conventional processes and the new method. Detailed investigations into individual factors that might impact cell viability results, such as cooling rates, warming rates, composition and properties of the cryoprotectant, are left for future efforts.
In this research, we compared the post-thaw cell conditions from two widely used conventional protocols, with those from the BioFlash Drive™ SP protocol. Cell lines tested in this research include Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™).
2.3. Sample Preparation The following sample preparation method is applied to all tested cell lines and in all cryopreservation methods in this research: Prepare the culture medium by mixing DMEM (Invitrogen, Carlsbad, CA) and Fetal Bovine Serum (FBS) (Invitrogen) at a 9:1 ratio. Culture the cell lines with the culture medium above using petri dishes, in 5% CO2 incubators at 37℃. Remove the culture medium.Wash the adherent cells in the petri dishes with PBS (Invitrogen) twice. Dispense 300µl 0.25% trypsin into each petri dish and place the petri dishes in the incubator (5% CO2, 37℃) for 1min.Take out the petri dishes and suspend the cells in each petri dish using 5ml PBS, and transfer the cell suspension to a 15ml centrifuge tube. Centrifuge the centrifuge tube, and remove the supernatant.Resuspend the cells with 6ml PBS, conduct cell viability assay on the sample to obtain total cell count X at this moment.If X is equal to or higher than 4×107, proceed with the next step; if X is lower than 4×107, new cell samples have to be prepared to make sure we have enough cells for this experiment.Transfer 6 ml cell suspension to a centrifuge tube. Label this tube as Tube 1. Transfer 3 ml cell suspension from Tube 1 to another centrifuge tube, labeling it as Tube 2. Centrifuge Tube 1 and Tube 2, and remove the supernatant. 2.4. Freezing for Experiment Group (Tube 1) Resuspend the cell pellet using FBS, which has been precooled to 4℃ beforehand, to give a final cell concentration between 1-2 x 106
cells/ml.Conduct cell viability assay on the sample in Tube 1, record the cell concentration, cell viability, and total cell count.Divide nine Fibulas™ BioFlash Drive™ SP in to three groups (group C, D, E) of three, and label them as C1-C3, D1-D3, E1-E3, respectively. Each group is subject to different test conditions, and the three vials within the same group give a repetition of three times.Transfer 1ml cell suspension from Tube 1 to each BioFlash Drive™ SP vial, and seal the cap.Within 3 minutes after adding samples into the vials, place vials of group C and E into a 5 x 5 cardboard box and place the box into a -80℃ freezer overnight. Subsequently, transfer the cardboard box containing group C and E to liquid nitrogen tank for storage. Samples are not immersed into or in direct contact with liquid nitrogen. (group E will be cultured without washing the cryoprotectant, while group C will be cultured after washing away the cryoprotectant).Place vials of group D into a 5 x 5 cardboard box and place the box in the gas phase section of a liquid nitrogen tank close to the liquid surface. Samples are not immersed into or in direct contact with liquid nitrogen.
2.5. Freezing for Control Group (Conventional methods, Tube 2) Resuspend the cell pellet using 10% v/v DMSO (Sigma-Aldrich) in FBS (Invitrogen) solution, which has been precooled to 4℃ beforehand, to give a final cell concentration between 1-2 x 106 cells/ml.Conduct cell viability assay on the sample in Tube 2, record the cell concentration, cell viability, and total cell count.Divide six Corning 2ml internal threaded cryogenic vials in to two groups (group A, B) of three, and label them as A1-A3 and B1-B3, respectively. The three vials within the same group give a repetition of three times.Transfer 2ml cell suspension from Tube 2 to each cryogenic vial, and seal the cap.Within 3 minutes after adding
samples in to the vials, place vials of group A to a slow-freezing container (Mr. Frosty, Thermo Fisher) prefilled with IPA (Isopropanol, Sigma-Aldrich) according to the product instruction; place vials of group B to another slow-freezing container (CoolCell, Biocision) according to the product instruction.Place both slow-freezing containers to into a 80℃ freezer overnight.Transfer vials of both group A and B to a liquid nitrogen tank for storage. Samples are not immersed into or in direct contact with liquid nitrogen.
2.6. Thawing Method The following thawing method is applied to all tested cell lines and in all cryopreservation methods in this research. Warm the culture medium using 37℃ water bath. After storing in a liquid nitrogen tank for at least 72 hours, take the sample out carefully and warm it by using the water bath. Group A, B,C,and D: Transfer the content of each vial into a centrifuge tube and centrifuge it at 108g for 5 min at 4℃. Discard the supernatant. Suspend the cell with 1ml warm culture medium, and then transfer the cell suspension to one unused well in a 6-well culture plate (Corning™ Costar™ Flat Bottom Cell Culture Plates). Label this well
hours. Repeat the cell viability assay for Group b after culturing for 24 hours.
2.7. Cell Viability Assay The following cell viability assay is applied to all tested cell lines and in both cryopreservation methods compared in this research. In this research, Countess II (Invitrogen) was used for automated cell counting. The machine was in default setting and has been validated by comparison to flow cytometry tests for providing satisfactory cell counting results to our lab when counting Vero (ATCC® CCL-81™) and HEp-2 (ATCC® CCL-23™), two of the most frequently used cell lines in our lab. We preferred Countess II to manual methods because it provides less countto-count variability. Remove the medium from each culture well to be measured. Dispense 300µl 0.25% trypsin into each culture well and place the culture plates in the incubator (5% CO2, 37℃) for 1min. Take out the culture plates and suspend the cells in each culture well using 0.5ml PBS. For each culture well, mix 10ul 0.4% Trypan Blue with 10ul cell suspension from the culture well and add the sample into a piece of a commercial cell viability slide (Countess II). Insert the slide into a Countess II automated cell counter (Thermo Fisher). Record the cell count number as well as viability, calculate and normalize cell counts in different sample vials: Recovery %=(total cells in a culture well /derived total cells in the corresponding culture well before cryopreservation*) × 100% Viability %=alive cells in a culture well/total cells in a culture well × 100% Cell proliferation %=alive cells (24hr after thawing) in a Group b well/alive cells (3hr after thawing) in a Group a well × 100%
*Because the cells from one vial were divided into two culture wells, the total cells of one vial (before cryopreservation) was divided by 2 to represent the initial total cells in each culture well.
2.8. Statistical analysis Statistical analysis of data was performed using Prism 7 (Graphpad). Wilcoxon signed-rank test was performed because the distribution of the data was unknown, and also because the sample size was relatively small (n=3). P values of less than 0.05 were considered statistically significant.
3. Results 3.1. Experiment group and control groups have comparable performance, after being placed in -80
environment and then
transferred to -150 . Results of group A, B, and C show the performance of two conventional methods and the new method. All samples were placed into -80 prior to -150
freezer
storage, as shown in Fig.2. Fig.2a shows that there is not
significant difference among A, B, and C in terms of cell recovery % 24 hours after thawing. The high value of cell viability % of all groups in Fig.2b indicates that there is no obvious damage to the cell samples. Fig.2c shows cells in Group C has a much higher proliferation % than the control Groups, but no significant difference. 3.2. Both using the new protocol with BioFlash Drive™ SP, placing samples directly in -150 has comparable performance to placing samples in -80
environment and then transferring into -150
environment. As shown in Fig.3a, cells in group C and D do not have significant difference in terms of recovery %. Also, in Fig.3b, the high viability % in
both groups indicates that there is no obvious damage to the cell samples. In Fig.3c, the proliferation % of group C and D do not have significant difference for in each cell type. 3.3. The presence of cryoprotectant preloaded in BioFlash Drive™ SP in cell culturing has different impacts on different cell types. The experiment group E was tested to evaluate the impact of the cryoprotective substances remaining the cell suspension in BioFlash Drive™ SP after thawing. As shown in Fig.4a, cells in group C and E do not have significant difference in terms of recovery %. Also, in Fig.4b, the high viability % in both groups indicates that there is no obvious damage to the cell samples. In addition, as shown in Fig. 4c, Hep-2 cells in group E has a much lower proliferation than cells in group C, indicating different cell types might have different preferences to the cryoprotectant. 3.4. Different methods take different amount of time and steps to complete the cryopreservation process. Based on the methods used in this experiment, we compare the time and steps between the conventional freezing processes and the one with BioFlash Drive™ SP (as shown in the table 1). BioFlash Drive™ SP integrates the cryoprotectant and slow-freezing function into the vial itself. By eliminating the steps of mixing cryoprotectant, transferring samples in and out of slow-freezers, the overall time span for cryopreservation is compressed from across 2 days to about 60 seconds. Although it does not seem that this new method fundamentally changes the principle of slow-freezing, it greatly reduces the time and steps needed to complete the process, making it more convenient to store and archive samples in different research stages. 4. Discussion
According to the results above, there are no statistically significant differences between the conventional protocols and the simplified protocol using BioFlash Drive™ SP in cryopreserving HEp-2 and Vero (Fig.2.). While both using BioFlash Drive™ SP , there is no significant difference of recovery % and viability % between samples first placed in -80
environment and then transferred to -150 , and those directly
placed in -150
environment (Fig.3). This observation should be further
investigated and verified by experimenting with more type of cells. Also, the presence of cryoprotectant from BioFlash Drive™ SP in post-thaw culturing is shown to have different impacts on different cell types. This is a topic worth further investigation.
BioFlash Drive™ SP introduces a new and simplified protocol for cryopreservation(Table 1). With this integrated solution vial, users are able to cryopreserve samples without slow-freezing containers, controlled-rate freezers, or pre-mixing of freezing medium. The results show decent performance on Vero and HEp-2 cells. Although BioFlash Drive™ SP does not show significant difference in results compared to conventional methods, it takes less time, and requires less steps, which make it more convenient for researchers to archive cell samples during experiment process. It may add value to researchers who want to save their samples often.
As mentioned before, due to constrains in time and resources, we were not able to go into details and decompose how BioFlash Drive™ SP achieve similar results as the conventional methods. Although it is claimed to have a thermal insulative layer, which helps it to achieve a 1 /min of cooling curve, it is not verified within this study. Also, the product also claims to have control-release for its cryoprotectant, yet
limited by the scope of this study, we have not studied the concentration change of cryoprotectant over time. We think these are all interesting questions to look into in the future.
5. Conclusion There are no statistically significant differences between the conventional protocols and the simplified protocol using BioFlash Drive™ SP in cryopreserving HEp-2 and Vero. Moreover, using BioFlash Drive™ SP might generate some extra benefits if users choose to place the samples directly into -150 , because this shortcut will not be possible when using conventional methods, which usually require samples to stay in -80 for some time first. Yet the results show that BioFlash Drive™ SP's cryoprotective substances will have different impacts on different cell types. Therefore, users might need to use discretion when using this new product.
This research is important because, to our knowledge, there is no existing study on this new and integrated design for cryopreservation. This research
studied
the
efficiency
and
performance
of
different
cryopreservation methods, both conventional and new, helping researchers to gain insights into different practices, so that they can better choose protocols suitable for their needs.
Acknowledgements: This study was supported by grants from National Key Research and Development Program of China (2018YFC1200100), Entrepreneurship Leadership Project in Guangzhou Development Zone of China(CY2018003), the National Natural Science Foundation of China (NSFC 31570163), the Youth Project of State Key Laboratory of Respiratory
Disease (SKLRD-QN-201713), Guangdong Medical Science and Technology Research Center Project (A2019460) and Scientific Research Project of Guangdong Province Traditional Chinese Medicine Bureau (20201191)
Author Contributions: T.Liu and R.Zhou：designed the experiments and wrote the paper. D.Xu: conducted the experiments. All experiments were performed at State Key Laboratory of Respiratory Disease in Guangzhou Medical University, China. Fibulas did not participate in any experiments.
Conflicts of interest The authors declare that they have no conflicts of interest.
References: [1] ATCC Animal Cell Culture Guide. https://www.atcc.org/~/media/PDFs/Culture Guides/AnimCellCulture_Guide.ashx. (2014). [2] J.M. Baust, W.L. Corwin, R. VanBuskirk, and J.G. Baust, Biobanking: The Future of Cell Preservation Strategies. Adv Exp Med Biol 864 (2015) 37-53. [3] J.G. Day, and M.R. McLellan, Cryopreservation and Freeze-Drying Protocols Volume 38 || Cryopreservation of Algae. 10.1385/0896032965 81-90. [4] J. Farrant, H. Lee, and C.A. Walter, Effects of Interactions Between Cooling and Rewarming Conditions on Survival of Cells. Ciba Found Symp 52 (1977) 4967. [5] R.J. Hay, Preservation of cell-culture stocks in liquid nitrogen. Tca Manual 4 (1978) 787-790. [6] R.J. Hay, Caputo, J., & Macy, M. L. , Preservation of cell cultures in liquid nitrogen. ATCC Quality Control Methods for Cell Lines. (1992). [7] R. Li, R. Johnson, G. Yu, D.H. McKenna, and A. Hubel, Preservation of cellbased immunotherapies for clinical trials. Cytotherapy 21 (2019) 943-957. [8] J.E. Lovelock, The haemolysis of human red blood-cells by freezing and thawing. Biochim Biophys Acta 10 (1953) 414-426. [9] B.J. Luyet, and P.M. Gehenio, Life And Death At Low Temperatures. 3 (1940) 33-99. [10] Mazur, and P., Kinetics of Water Loss from Cells at Subzero Temperatures and the Likelihood of Intracellular Freezing. Journal of General Physiology 47 347-369.
[11] C.A.V. Polge, A.U. Smith, and A.S. Parkes, Revival of Spermatozoa After Vitrification on and Dehydration at Low Temperatures. Nature 164 (1949) 666. [12] M.J. Ryan, and D. Smith, Cryopreservation and freeze-drying of fungi employing centrifugal and shelf freeze-drying. Methods Mol Biol 368 (2007) 127-40. [13] M.J. Taylor, and D.E. Pegg, The effect of ice formation on the function of smooth muscle tissue stored at -21 or -60 °C. 20 0-40. [14] G. Vunjaknovakovic, and R.I. Freshney, Culture of Animal Cells - A Manual of Basic Technique and Specialized Applications, 7th Edition, 2016.
Fig .1. A individual unit of BioFlash Drive™ SP. Fig.2. Post-thaw results of group A, B, and C: 2a, Recovery % 24hours after thawing; 2b, Viability % 24hours after thawing; 2c, Cell Proliferation %. Sample Label: A: IPA box B: foam box C: meta sp-80. Note:Group A and B are control groups of conventional methods; group A uses IPA slow-freezing box while group B uses foam slow-freezing box. Group C and D are experiment groups of the kit, where group C is placed in -80 , group D in -150 . Fig.3. Post-thaw results of group C, and D: 3a, Recovery % 24hours after thawing; 3b, Viability % 24hours after thawing; 3c, Cell Proliferation %. Sample Label: C: meta sp-80 D: meta sp-150 Note: Group C and E are experiment groups of the kit, where group C is placed in -80 . Group E is the only group that is placed in -80
while
the sample within is cultured with the cryoprotectant after thawing. Fig.4. Post-thaw results of group C and E: 4a, Recovery % 24hours after thawing; 4b, Viability % 24hours after thawing; 4c, Cell Proliferation %. Sample Label: C: meta sp-80
E: meta sp-80 cpa
Table 1 Comparison of Two Cell Freezing Protocols Step
Conventional Freezing Process
BioFlash Drive™ SP
1
Mix cryoprotectant
----------
2
Transfer cell suspension to vials
Transfer cell suspension to BioFlash Drive™
3
Prepare a slow-freezing container ---------and slowly freeze samples in -80 . Keep samples there overnight.
4
Take
samples
out
from
slow- ----------
freezing containers 5
Total
Transfer samples to freezers or Put samples directly to -80 freezers or liquid nitrogen tanks
vapor phase nitrogen
Across 2 days
About 60 seconds
Highlights and eTOC Blurb Highlights : The present research compares a novel cell cryopreservation kit and conventional cell freezing methods. The results show that there is no significant difference among these methods. The performance of the kit under non-usual extreme conditions is also studied.