Human serum albumin-containing xeno-free freezing medium optimized for regenerative medicine

Abstract

Human-induced pluripotent stem (iPS) cells are expected to be used in regenerative medicine and drug toxicity tests. Earlier, human iPS cells were cryopreserved by the vitrification method because the slow freezing of human iPS cells resulted in low recovery following thawing. However, the vitrification method requires a high level of skill to rapidly prepare many frozen cell stocks. Furthermore, a dry shipper is necessary to transport the stocks of vitrified human iPS cells because simple transport of vitrified cell stocks on dry ice was impossible. Transportation using dry shippers is logistically cumbersome and expensive. In this study, we developed a new slow-freezing medium to prepare cell stocks that can be transported on dry ice without a dry shipper. This medium allowed the preparation of several stocks of frozen cells of the same lot simultaneously and their transportation on dry ice. Using human peripheral blood mononuclear cells (PBMCs) and human iPS cells, we investigated the optimal slow preservation solution. We found that a slow freezing medium containing 10% (v/v) dimethyl sulfoxide (DMSO) and 1% (v/v) human serum albumin (HSA) was optimal for cryopreservation of human PBMCs and iPS cells. This xeno-free freezing medium was shown to have the potential for application in the cryopreservation of cells used in regenerative medicine.

Highlights

1. A xeno-free freezing medium was developed to enable highly efficient cryopreservation of human PBMCs and iPS cells using the slow freezing method.

2. This freezing medium is easy to use.

3. Cell stock cryopreserved in this freezing medium can be transported on dry ice.

4. This freezing medium represents a potentially useful reagent for regenerative medicine.

Introduction

Cell-based immunotherapies, such as adoptive cytotoxic T, natural killer, and dendritic cell transfer, are effective for some tumors [1,2,3,4]. These techniques require patient-derived peripheral blood mononuclear cells (PBMCs) to be cryopreserved as stocks, which can then be thawed when necessary. Processes, including cryopreservation, storage, transportation, and thawing, require high recovery rates after thawing owing to the limited availability of patient-derived PBMCs.

Human embryonic stem (ES)/induced pluripotent stem (iPS) cells are expected to be used in regenerative medicine [5], drug screening [6], model systems for human biology [7], and human virus infection models [8]. Human ES/iPS cells are cultured, maintained, and frozen under specific conditions, in contrast to murine ES/iPS cells [9]. In addition, it is essential to transport these cells from the location of storage of the cell stocks to the users as cryopreserved cells without any loss during transport. It was reported that the viability of human ES/iPS cells was significantly lower after thawing by the usual slow freezing method compared to the other general somatic cell lines [9], suggesting that human ES/iPS cells have low freeze resistance. The vitrification method has the advantage of cryopreserving rodent early stage embryos compared to standard programmed slow cryopreservation in freezing medium containing dimethyl sulfoxide (DMSO) [10], and it has also been applied to fertilized eggs and early stage embryos of cows, pigs, and sheep [11,12,13,14]. This method involves the direct immersion of cells in liquid nitrogen using a solution containing a mixture of high-concentration DMSO, acetamide, or propylene glycol among others, and rapid cooling to freeze the water in a vitrified state without crystallizing it [15]. However, some components of the vitrification solution are toxic to cells because they exert high osmotic pressure on the cells [9].

Slow freezing involves immersion of cells in a solution containing a cryoprotectant such as DMSO or glycerol. The cells are then frozen by cooling via lowering the temperature to −1 K/min [16]. The slow freezing method does not require rapid operation during freezing and thawing; in contrast, the vitrification method requires a high level of skill to rapidly prepare several frozen cell stocks. Furthermore, a dry shipper is necessary to transport vitrification-frozen human iPS cell stocks because dry ice conditions are inadequate for vitrified cells. Transportation using dry shippers is logistically cumbersome and expensive.

The objective of this study was to develop a slow-freezing medium for transportation of cells on dry ice. Cryopreservation of human PBMCs and human iPS cells that are used for regenerative medicine, which are harder to cryopreserve than the standard cell lines, such as NIH3T3-3 cell line, require special conditions especially in terms of the composition of the freezing medium.

In this study, we developed a cell-freezing medium that enables highly efficient cryopreservation of human PBMCs and iPS cells using the slow freezing method, which is easy to use and can be used for transportation of cells with dry ice.

Materials and Methods

Cells

Human PBMCs, obtained from Precision for Medicine (Frederick, MD, USA), were grown on OKT3 (Janssen, Tokyo, Japan) in RPMI 1640 + 9S Sin medium (GC Lymphotec, Tokyo, Japan) to maintain the cells in an activated state [17]. The human iPS cell line 1231A3, obtained from RIKEN Bioresource Center (Tsukuba, Japan), was grown on an iMatrix-511 (Matrixome, Osaka, Japan) layer in StemFit AK02N medium (Takara Bio, Kusatsu, Japan) to maintain the cells in an undifferentiated state [18]. NIH3T3-3 (RCB0150), obtained from RIKEN Bioresource Center, was grown in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Gibco, Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS; Biosera, Kansas City, MO, USA). PBMCs, undifferentiated human iPS 1231A3 cells, and NIH3T3-3 cells were cryopreserved and stored in Bambanker freezing medium (GC Lymphotec) prior to this study.

Slow-freezing and thawing of cells

NIH3T3-3 cells were dissociated from the culture dish and centrifuged at 190 × g for 3 min. The cell pellets were resuspended in 1 ml (5 × 10⁵ cells) of pre-chilled freezing medium, and then immediately transferred to a cryotube and placed into BICELL (Nihon Freezer Co., Ltd., Tokyo, Japan) for slow freezing overnight at −80°C. After overnight incubation, the cryotube was transferred to liquid nitrogen and stored until use. The freezing media were prepared as follows: 0 to 20% (v/v) DMSO (Nacalai Tesque, Kyoto, Japan), 10% (v/v) sucrose (Sigma-Aldrich, St. Louis, MO, USA), or 10% glucose (Wako, Tokyo, Japan) in DMEM with high glucose containing 10% (v/v) FBS.

Each freezing medium was prepared as follows: 0 to 30% (v/v) DMSO (Nacalai Tesque) and 0 to 2.5% (v/v) human serum albumin (HSA: CSL Behring, Tokyo, Japan) in the basic freezing medium supplied by GC Lymphotec Inc. [19]. PBMCs were activated, cultured in culture flasks, and centrifuged at 190 × g for 3 min. The cell pellets were resuspended in 1 ml (2.5 × 10⁶ cells) of pre-chilled freezing medium, immediately transferred to a cryotube, and placed into BICELL for slow freezing in a −80°C freezer overnight. After overnight incubation, the cryotube was transferred to liquid nitrogen and stored until use.

Human iPS cells were dissociated from the cells on the iMatrix-511 coated plastic dish with 0.5 × TrypLE™ Select CTS™ (Thermo Fisher Scientific, New York, NY, USA). Following counting of the cells, the human iPS cell suspension was centrifuged at 190 × g for 3 min to obtain the pellet, which was resuspended (1.4 × 10⁶ cells/ml) in pre-chilled freezing medium. The cell suspension was immediately transferred to a cryotube and placed into a BICELL for slow freezing in a −80°C freezer overnight. After overnight incubation, the cryotube was transferred to liquid nitrogen and stored until use.

For thawing, the frozen tubes were incubated at 37°C in a water bath for 1 min, and then the thawed cell suspension was transferred to 7 ml of culture medium. This suspension was centrifuged at 190 × g for 3 min to obtain a pellet, which was resuspended in culture medium. Cell viability was determined by staining the dead cells with 0.4% Trypan Blue stain (Thermo Fisher Scientific) and counting the live cells with a hemocytometer TC20 (Bio-Rad, Tokyo, Japan). The cells were plated in an adequately sized culture plate.

Vitrification freezing and rapid thawing of cells

Human iPS cells were dissociated from the cells on the iMatrix-511 coated plastic dish with 0.5 × TrypLE™ Select CTS™. Following counting of the cells, the human iPS cell suspension was centrifuged at 190 × g for 3 min to obtain a pellet, which was resuspended (1.4 × 10⁶ cells) in 0.2 ml of pre-chilled freezing medium for human ES/iPS cells (DAP213:2 M DMSO, 1 M acetamide, 3 M propylene glycol) (RiproCELL, Yokohama, Japan). The cell suspensions were transferred immediately to a cryotube, the tubes were immersed in liquid nitrogen solution within 20 sec, and stored until use.

For thawing, pre-warmed (37°C) culture medium was directly added to the frozen tubes, and then the thawed cell suspension was transferred to an excess amount (7 ml) of culture medium. This suspension was centrifuged at 190 × g for 3 min to obtain a cell pellet, which was then resuspended in culture medium. Cell viability was determined by staining the dead cells with 0.4% Trypan Blue and counting the live cells with TC20 after resuspension in culture medium. The cells were plated in an adequately sized culture plate.

Alkaline phosphatase (ALP) staining

ALP activity was visualized using Stemgent Alkaline Phosphatase Staining Kit II (ReproCELL) according to the manufacturer’s instructions.

Statistical analysis

The significance of the differences between groups was determined using a paired t-test and the statistical processing software GraphPad Prism 8 J (GraphPad Software, San Diego, CA, USA).

RESULTS

Optimization of HSA and DMSO concentrations in freezing medium using human PBMCs

First, we determined the adequate concentration of DMSO and other supplements, such as sucrose or glucose, in slow-freezing medium for cryopreservation of stocks of NIH3T3-3, a standard cell line. The slow-frozen cells were thawed and cultured for 1 day. The number of viable cells was counted using the trypan blue exclusion method. As shown in Fig. 1, the optimum condition for NIH3T3-3 cells was 10% DMSO (v/v), suggesting that the recovery of frozen cells depends on the conditions of cryopreservation.

Fig. 1.

Effect of composition of freezing medium on post-thaw viability and recovery of NIH3T3-3 cells. Cell viability after cryopreservation in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. Data are shown as means ± standard deviation (SD), n=3, *P<0.05, **P<0.01.

IL-2 and anti-CD3 antibody-activated human PBMCs were frozen in freezing media containing 0–2.5% HSA or 0–30% DMSO. Regarding HSA concentration, each freezing medium contained 0–2.5% HSA (v/v) with 10% DMSO (v/v) (Fig. 2A). The frozen cells were thawed and cultured for 5 days. The number of viable cells was counted using the same method as described above. 1% HSA (v/v) was found to be optimal for the recovery of cells under the condition of 10% DMSO.

Fig. 2.

Selection of freezing medium for slow-freezing of human peripheral blood mononuclear cells (PBMCs). (A) Effects of human serum albumin (HSA) concentration on cryopreservation efficacy of freezing media. Each freezing medium contained 0, 0.5, 1.0, 1.5, 2.0 or 2.5% HSA (v/v). Human activated PBMCs were cryopreserved for 14 days using six different concentrations of HSA. The cells were thawed, resuspended in culture medium, and cultured, and the viable cell count was determined by trypan blue staining on the day 5th (n=3). (B) Effects of dimethyl sulfoxide (DMSO) concentration on cryopreservation efficacy of freezing media. Each freezing medium contained 0, 5, 10, 15, 20, 25, or 30% DMSO (v/v). Human PBMCs were cryopreserved for 14 days using seven different concentrations of DMSO. The cells were thawed, resuspended in culture medium, and cultured. Viable cell counts were determined by trypan blue staining (n=3). Data are shown as means ± SD, n=3, *P<0.05, **P<0.01, ***P<0.005.

Next, regarding the DMSO concentration, each freezing medium contained 0−30% DMSO (v/v) with 1% HSA (v/v) (Fig. 2B). The frozen cells were thawed and cultured for 6 days. The number of viable cells was counted using the trypan blue exclusion method. 10% DMSO (v/v) was found to be optimal for cell recovery.

These results suggests that freezing medium containing 10% DMSO (v/v) and 1% HSA (v/v) is optimum for human PBMCs.

Comparison of optimum slow freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) and vitrified freezing medium using human iPS cells

The cryopreservation efficiency of human iPS cells in optimized slow freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) was compared to that in DAP213, which is a commonly used vitrification freezing medium. After cryopreservation in liquid nitrogen for 13 days, human iPS cells cryopreserved in either freezing medium were thawed and then cultured on an iMatrix-511-coated plastic plate. Analysis of cell viability on the 6th day of culture following thawing revealed no significant differences in either cell viability or cell repopulation capability between the different cryopreservation media (Fig. 3A).

Fig. 3.

Comparison of optimized slow freezing and vitrified cryopreservation media. (A) Effect of the optimized slow freezing medium was compared with that of the vitrified freezing medium. Human-induced pluripotent stem (iPS) cells 1231A3 were cryopreserved for 8 days using optimized slow cryopreservation solution (10% dimethyl sulfoxide (DMSO) [v/v], 1% human serum albumin (HSA) [v/v]) and DAP213 (2M DMSO/1M Acetamide/3M Propylene glycol). Human iPS cells 1231A3 were resuspended in culture medium and cultured, and viable cells were counted using trypan blue staining on day 7 (n=3). (B) Image of cells frozen with optimized slow cryopreservation solution (10% DMSO [v/v], 1% HSA [v/v]) on the 4th day of thawing. (C) Image of cells frozen with DAP213 on the 4th day of thawing. (D) Alkaline phosphatase (ALP) staining of human iPS cells 1231A3 that were frozen in optimized slow cryopreservation solution (10% DMSO [v/v], 1% HSA [v/v]), thawed, and maintained for 7 days after thawing. (E) ALP staining of human iPS cells 1231A3 that were frozen in DAP213, thawed, and maintained for 7 days after thawing. Data are shown as means ± SD, n=3. N.S.; not significant.

Each frozen stock of human iPS cells was thawed and cultured in plastic dishes for 6 days. Morphological analysis of the human iPS cell colonies and ALP staining experiments indicated that the human iPS cells proliferated on the 6th day of culture as undifferentiated cells (Fig. 3B–E). These data suggest that human iPS cells can be cryopreserved and thawed satisfactorily using this freezing medium.

Comparison of 48 hr dry ice transport of slow freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v), and vitrified freezing medium using human iPS cells

Furthermore, the cell proliferation efficiency after 48 hr of transport on dry ice was examined using freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) and vitrified freezing medium. Human iPS cells cryopreserved in liquid nitrogen for 13 days in either of the freezing media were transported on dry ice for 48 hr. After transport, frozen cells were thawed and cultured on an iMatrix-511-coated plastic plate. The cells were collected on the 7th day of culture, and the number of viable cells was counted. The number of live cells cryopreserved in the freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) was higher than that in DAP213 (P<0.01) (Fig. 4A). ALP staining was performed to confirm that the human iPS cells were in an undifferentiated state. Following thawing from the frozen medium containing 1% HSA (v/v) and 10% DMSO (v/v), the human iPS cells growing on the 6th day of culture were confirmed to be undifferentiated cells. (Fig. 4B–E). These data suggest that human iPS cells can be transported using dry ice using this slow-freezing medium.

Fig. 4.

Comparison of optimized slow- and vitrified freezing media for dry ice transportation. (A) Preservation effect of optimized slow- and vitrified freezing media on induced pluripotent stem (iPS) cells that were transported on dry ice for 48 hr. iPS cells 1231A3 were cryopreserved for 8 days using optimized slow freezing medium or DAP213 and transported on dry ice for 48 hr. Human iPS cells were then thawed, resuspended in culture medium, and cultured, and viable cell counts was determined by trypan blue staining on the day 7. Experiments were performed in triplicate (n=3). **P<0.01. (B) Image of cells frozen with optimized slow freezing medium on the day 4 of culture. (C) Image of cells frozen with DAP213 on day 4 of culture. (D) Alkaline phosphatase (ALP) staining of human iPS cells 1231A3 that were frozen in optimized slow freezing medium, thawed and cultured for 7 days after thawing. (E) ALP staining of human iPS cells 1231A3 that were frozen in DAP213, thawed, and cultured for 7 days after thawing. Data are shown as the mean ± SD, n=3, **P<0.01.

Discussion

The freezing medium used for cryopreservation of cells administered to humans for regenerative medicine must be xeno-free. Several xeno-free freezing media have been previously reported and include the following: a vitrification freezing medium containing HSA for hES cells [15], a slow freezing medium containing DMSO, anhydrous dextrose, and polymer for hES/iPS cells [20], and a xeno-free commercial slow freezing medium for adipose-derived mesenchymal stem cells [21]. The slow freezing media are less cytotoxic [9] and easier to use [9] than the vitrification freezing medium, and can be used to process large number of cells simultaneously [9]. Therefore, slow freezing media are considered to be more beneficial for regenerative medicine. There are many slow-freezing media containing DMSO and FBS for general cell lines. DMSO and FBS protect against freezing damage. However, it is challenging to use FBS in a slow-freezing medium for regenerative medicine because FBS contains many unknown bovine-derived components. In contrast, the main component of FBS is albumin, which has a cryoprotective effect.

Therefore, we focused on DMSO and HSA, which act as cryoprotectants in the main freezing medium, and investigated a new cryopreservation solution for regenerative medicine. The slow freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) was found to be optimal for human PBMCs (Fig. 2A, 2B). The optimized slow freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) showed the same cryopreservation effect in human iPS cells as the vitrification medium (Fig. 3A). Furthermore, after 48 hr of transport of human iPS cells on dry ice, the optimized slow freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) was shown to have a much better cryopreservation effect than the vitrification medium (Fig. 3B).

The freezing medium containing 1% HSA (v/v) and 10% DMSO (v/v) has a significant advantage over DAP213 with respect to transportation of cells (Fig. 4A). Cells frozen by the vitrification freezing method, such as that using DAP213, are in a supercooled state, and the intracellular water is in a vitrified state. However, under dry ice storage at approximately −80°C, which is higher than the glass transition temperature (approximately −130°C), ice crystals are formed inside the cells and the cells become damaged and die. In contrast, freezing in the slow freezing method does not result in vitrification of intracellular water; thus there are no ice crystals formed even under dry ice conditions even under dry ice conditions, where the temperature is higher than the glass transition temperature; therefore, there is little damage to cells.

Although several methods for freezing of single cells have been established, freezing methods for cell clumps such as spheroids, sheet-like cells, and cell tissues such as PDX have not yet been established and are a topic for future studies.

In conclusion, we demonstrate that our freezing medium is useful for effective, easy, safe, and economical cryopreservation and banking of clinical-grade human cell stocks that have applications in regenerative medicine.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This study was supported by a Grant-in-Aid for Challenging Exploratory Research (No. 24650254) and Scientific Research (A) (No. 25242040) from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific Research on Innovative Areas (No. 231190003) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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