Cryopreservation in Situ of Cell Monolayers on Collagen Vitrigel Membrane Culture Substrata: Ready-to-Use Preparation of Primary Hepatocytes and ES Cells

Abstract

Cryopreservation is generally performed on cells in suspension. In the case of adherent cells such as hepatocytes, a loss of their ability to attach is a more serious problem than a decreased viability after cryopreservation. We herein report a novel technology of direct in situ cryopreservation of cells cultured on collagen vitrigel membranes, which have excellent mechanical strength and can be easily handled by tweezers even when coated with cultured cells. Rat primary hepatocytes, mitomycin C-treated mouse fibroblasts (feeder cells for ES cells), and mouse ES cells on the feeder cells were cultured on collagen vitrigel membranes for 1 day. The membranes with cells attached were then plucked up from the dish, soaked in cryopreservation medium containing 10% dimethyl sulfoxide, frozen using a controlled-rate freezer, and transferred to liquid nitrogen. The cells cultured on plastic cell culture dishes were also frozen as controls. After storage in liquid nitrogen for periods from 1 week to 3 months, the cryopreserved membranes with the cells still attached were thawed by adding warmed culture medium. Cell viability estimated by morphology and functional staining with calcein showed significant improvement in comparison to cells cryopreserved without the collagen vitrigel membrane. The recoveries of living cells after cryopreservation were 26.7%, 76.2%, and 58.6% for rat hepatocytes, mitomycin C-treated mouse fibroblasts, and mouse ES cells on collagen vitrigel membranes, respectively. In contrast, essentially no cells at all remained on the plastic cell culture dishes after thawing. Because adherent cell storage under these conditions is very convenient, the use of this technique employing collagen vitrigel membranes should be generally applicable to the cryopreservation of adherent cells that are otherwise problematic to store as frozen stocks.

Introduction

The preservation of cells is a key technology not only for research in cell biology but also for clinical cell transplantation. Although a number of preservation protocols have been proposed, the cryopreservation of adherent cells such as primary hepatocytes and primate ES cells still remains difficult. There are several important factors for obtaining the successful cryopreservation of cells; for example, the composition of the medium and cryoprotectants, and the freezing and thawing procedures (5,7,10,12). Although many investigations have attempted to achieve improvements in cryopreservation solutions using isolated cells in suspension, there are fewer studies on freezing adhering cells. The collagen vitrigel membrane, a new type of collagen scaffold, is a highly dense stable gel membrane prepared by the three processes of gelation, vitrification, and rehydration, as previously described (16). We herein report the successful in situ cryopreservation of adherent cells cultured on collagen vitrigel membranes. We cultured three types of cells—primary rat hepatocytes, mitomycin C-treated primary mouse embryonic fibroblasts (R-PMEF-N), and mouse embryonic stem (ES) cells—on the collagen vitrigel scaffold on which such cells spread and formed a monolayer. The present method showed a marked superiority in the recovery of these cells in comparison to the standard methods.

Materials and Methods

Materials

The materials used in the present study were Dulbecco' modified Eagle's medium (DMEM), fetal bovine serum for ES cells, antibiotics (penicillin, streptomycin, and amphotericin B), and MEM nonessential amino acids solution, all from Gibco BRL, Life Technologies (Grand Island, NY). Fetal bovine serum for hepatocytes was from Gemini Bio-Products (West Sacramento, CA). Nucleosides (adenosine, cytidine, guanosine, and uridine), 2-mercaptoethanol (M6250), and dimethyl sulfoxide (DMSO, D2650) were from Sigma-Aldrich (St. Louis, MO). ESGRO (Murine LIF) (ESG1107) was from Chemicon International (Temecula, CA). Live/Dead Viability Cytotoxicity Kit (L3224) was from Molecular Probes (Eugene, OR). All other materials and chemicals not specified above were of the highest grade available.

Animals and Cells

Male Sprague-Dawley rats (6–7 weeks old, specific pathogen free) weighing approximately 200 g were purchased from Sankyo Lab (Tokyo, Japan). Mouse ES cell (R-CMTI-1A; mouse embryonic stem cell, 129 line at passage 13) and primary mouse embryo neomycin-resistant and mitomycin C-treated fibroblasts (R-PMEF-N) were purchased from Dainippon Sumitomo Pharma (Osaka, Japan).

Preparation of Collagen Vitrigel

The collagen vitrigel membrane was prepared using type I collagen (Cellgen I-AC, Koken, Tokyo, Japan) as described previously (16). In some experiments, the vitrigel membrane was irradiated with ultraviolet light at a dose of 8,000 J/m² to cross-link the collagen fibers (19), using FUNA-UV-LINKER, FS-1500 (Funakoshi, Tokyo, Japan).

Isolation and Culture of Primary Rat Hepatocytes

The primary hepatocytes were isolated from SD rat livers using collagenase (S-I, NITTA Gelatin, Tokyo, Japan) as described previously (3). The viability of cells used was greater than 80%, as determined by the trypan blue dye exclusion test (Trypan Blue Stain, Gibco BRL). Freshly isolated hepatocytes (4 × 10⁵ cells) were inoculated onto collagen vitrigel membranes (35 mm in diameter) placed in 35-mm culture dishes and cultured in 2 ml of DMEM containing 20 mmol/L HEPES, 10 mmol/L nicotinamide, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal bovine serum. As controls, cells were cultured in 35-mm culture dishes without vitrigel (Falcon3001, Becton Dickinson, Franklin Lakes, NJ). The culture medium was changed at 3 and 6 h after starting the culture. Three hours after inoculation, the collagen vitrigel membrane was detached from the bottom of the dish to allow the culture medium to come into contact with the cells through the membrane. The cells were then incubated for 24 h in a 37°C incubator under a humidified 5% CO₂ atmosphere.

ES Cell and R-PMEF-N Feeder Cell Culture

A mouse ES cell culture was performed with mitomycin C-treated R-PMEF-N feeder cell layers as follows: first, 2 × 10⁵ of R-PMEF-N feeder cells was inoculated onto UV-irradiated collagen vitrigels with 2 ml of DMEM containing 10 mmol/L MEM nonessential amino acids solution, 30 μmol/L of adenosine, cytidine, guanosine, and uridine, 100 μmol/L 2-mercaptoethanol, 1,000 U/ml ESGRO, 100 U/ml penicillin, 100 U/ml streptomycin, 100 μg/ml amphotericin B, and 20% FBS. Twenty-four hours thereafter, 1 × 10⁶ of mouse ES cells were seeded onto the feeder cell layer. Three hours after the inoculation of feeder cells or ES cells, the collagen vitrigel membrane was detached from the bottom of the dish to allow the culture medium to come into contact with the cells through the membrane. The plates were incubated for 24 h in a 37°C incubator under a humidified 5% CO₂ atmosphere and then cryopreserved.

Cell Freezing and Thawing Procedures

Prior to freezing, the culture medium was replaced by medium containing 10% DMSO. The dishes were placed in a controlled rate freezer (Kryo10, Planer, Middlesex, UK) and frozen at a rate of 1°C/min as described previously (12). Immediately after freezing, the culture dishes were transferred to liquid nitrogen and stored until use (from 1 week to 3 months).

To thaw the cells, 2 ml of culture medium warmed to 37°C was added. Just after the last ice crystal disappeared, the medium was changed to fresh culture medium. The cells were cultured and observed at 1 and 6 h after thawing.

Assessment of Outcome of Cryopreservation

To assess the outcome of each procedure, the amount of live cells before and after cryopreservation was determined quantitatively by an image analysis. The cells on the dish or collagen vitrigel membrane were stained with a Live and Dead Viability Cytotoxicity Kit 3 h after thawing. To determine the cell viability of fibroblasts and hepatocytes, the total number of cells was counted under phase contrast microscopy and the number of dead cell nuclei stained by ethidium bromide was determined with fluorescence microscopy (area 0.35 mm², original magnification 100×). The number of dead hepatocytes was estimated as half the number of red-stained nuclei, because >95% of the hepatocytes were binuclear. Mouse ES cells form three-dimensional spheroids, so the total cell number was calculated from the occupancy area based on the supposed cell number, 1,560/0.35 mm². This was estimated from the number of cells harvested from a 35-mm diameter dish at confluence, as determined by an image analysis. All image analyses were performed using NIH Image software program from the National Institutes of Health. The value of each experimental group was obtained from three independent experiments as mean ± SD, and the value in each sample was the mean of three separate areas.

Statistical Analysis

Statistical significance was determined by one-way ANOVA and Fisher's Protected Least Significant Difference (PLSD) test using the StatView software program, version 5.0 for Macintosh (SAS Institute, Cary, NC).

Results

Cryopreserved Rat Hepatocytes on Collagen Vitrigel

One day after the inoculation, the primary rat hepatocytes adhered and spread well both on the plastic culture dishes and the collagen vitrigel membranes (Fig. 1A, B). The percentage of attached cells was 47.3 ± 2.9% and 62.6 ± 4.7%, respectively, as shown in Figure 1F. There were very few dead cells under both conditions (cell viability 96.6% and 98.0%, respectively). After cryopreservation and thawing, the cell morphology was retained well on the collagen vitrigel membrane (Fig. 1D), whereas almost no cells remained on the plastic culture dishes (Fig. 1C). The percentage of total attached cells and of viable attached cells was 31.5% and 16.4%, respectively, on collagen vitrigel membranes (Fig. 1E, F) (i.e., the viability of the recovered cells was 52.1%). We also froze isolated hepatocytes in suspension using the same cryopreservation medium as described above. Although the cell viability just after thawing was estimated at 40–60% by trypan blue dye exclusion, none of these hepatocytes attached to the plastic culture dishes (data not shown).

Figure 1.

Phase-contrast photomicrographs of rat hepatocytes before and after cryopreservation. Freshly isolated rat hepatocytes on plastic cell culture dish (A) and collagen vitrigel membrane (B), 24 h after inoculation. Hepatocytes on the dish after cryopreservation (C), and on collagen vitrigel membrane after cryopreservation (D). Cells on the dish or collagen vitrigel membrane had been stored in liquid nitrogen for 1 week to 3 months. (E) Fluorescence photomicrograph of (D), after staining with calcein and ethidium bromide. The bright green cells are viable. Scale bar: 100 μm. (F) The percentage of attached cells (open bars) and viable attached cells (dotted bars). The data are the means ± SDs (n = 3). Asterisks denote statistical significance (p < 0.05) by one-way ANOVA and Fisher PLSD.

Optimization of Mouse ES Cell Culture on Collagen Vitrigel Membranes

The mouse ES cells grew well in plastic culture dishes (Fig. 2A). However, the cell growth on the intact collagen vitrigel membrane was not satisfactory at first, because of detachment of the R-PMEF-N feeder cells (Fig. 2B). Because feeder fibroblasts prefer rigid scaffolding, the collagen fibers were cross-linked by ultraviolet irradiation of the intact collagen vitrigel membrane prior to seeding the feeder cells. Irradiation at doses of 2,000 or 8,000 J/m² (Fig. 2C, D), resulted in an increased amount of attached cells in a dose-dependent manner. Therefore, the collagen vitrigels were irradiated at 8,000 J/m² for the feeder and ES cell culture.

Figure 2.

Effect of UV irradiation of collagen vitrigel membrane on mouse ES cell and feeder cell culture. Mouse ES cell and feeder cell coculture on plastic cell culture dishes (A), nonirradiated collagen vitrigel membrane (B), UV-irradiated collagen vitrigel membrane at a dose of 2,000 J/m² (C) or 8,000 J/m² (D). Scale bar: 100 μm.

Cryopreservation of R-PMEF-N Feeder Cells and Mouse ES Cells on Collagen Vitrigel Membrane

The percentage of cells attaching to plastic dishes or vitrigel membranes was 43.2% and 44.5%, respectively, 24 h after inoculation (Fig. 3A, B, F). After cryopreservation, while no cells at all were recovered on the plastic dishes (Fig. 3C), 26.0% of the UV-irradiated collagen vitrigel membrane surface was covered with fibroblasts and 22.6% with live cells (Fig. 3D, E, F), so that total cell recovery was 58.6% after cryopreservation (Fig. 3F).

Figure 3.

Phase-contrast photomicrographs of mitomycin C-treated R-PMEF-N feeder cells before and after cryopreservation. R-PMEF-N cells on plastic cell culture dish (A) and collagen vitrigel membrane (B), 24 h after inoculation. R-PMEF-N cells on the dish after cryopreservation (C), or collagen vitrigel membrane after cryopreservation (D). Cells on the dish or collagen vitrigel membrane were stored in liquid nitrogen for 1 week to 3 months. (E) Fluorescence photomicrograph of (D), after staining with calcein and ethidium bromide (bright green indicates viability). Scale bar: 100 μm. (F) Percentage of attached cells (open bars) and viable attached cells (dotted bars). The data are the means ± SDs (n = 3). The asterisks denote statistical significance (p < 0.05).

The mouse ES cells grew as well with R-PMEF-N cells on the UV-irradiated collagen vitrigel membrane as on the plastic culture dishes (Fig. 4A, B). The percentage of ES cells attaching was 51.9% and 60.2%, respectively (Fig. 4F). After cryopreservation, the ES and feeder cells were completely detached from the plastic culture dishes (Fig. 4C) but of the cells cultured on the UV-irradiated collagen vitrigel membranes, the total cell and live cell recoveries were 44.1% and 39.1%, respectively (Fig. 4F). The ES cell viability after cryopreservation was 88.3% (Fig. 4D, E). In addition, the ES specific colony morphology was well retained 1 day after thawing (data not shown).

Figure 4.

Phase-contrast photomicrographs of ES cells and mitomycin C-treated R-PMEF-N feeder cells before and after cryopreservation. Cells cultured on plastic cell culture dish (A) and collagen vitrigel membrane (B). The details of culture schedule are as described in the text. The cells on the dish after cryopreservation (C), and on collagen vitrigel membrane after cryopreservation (D). The cells were stored in liquid nitrogen for 1 week to 3 months. (E) Fluorescence photomicrograph of (D), after staining with calcein and ethidium bromide (bright green indicates viability). Scale bar: 100 μm. (F) Percentage attached cells (open bars) and viable attached cells (dotted bars). The data are means ± SDs (n = 3). The asterisks denote statistical significance (p < 0.05).

Discussion

Collagen is a well-known cell matrix usually used as a thin coating material or gel material composed of a low density of collagen fibrils. Collagen vitrigel membranes composed of a high density of collagen fibrils possess excellent cell matrix features and structural strength (16). The latter trait facilitates the handling of the cells without destroying the cellular network formed during in vitro culture. In this report, we used the membrane for direct in situ preservation of primary hepatocytes, feeder cells, and ES cells. The cells on collagen vitrigel membranes are ready to use for experiments, such as drug testing and ES cell culture. Recently, mouse ES cells have been used for teratogenicity testing, by means of an approach known as embryonic stem cell testing (EST) (14). The availability of cryopreserved cultured ES cells on such membranes will be of great utility for well-standardized high throughput assays with strict quality control. In this regard, the present method is likely to be of interest to researchers in many different fields.

Cell encapsulation with biomaterials has been explored previously for cryopreserving cells. In 1990, Koebe reported that the cryopreservation of hepatocytes in two layers of collagen effectively increased the survival rate and the maintenance of function (9). Other gelatinous materials such as polyacrylamide gel and alginate gel have also been used (1,2,4,6,8,13,17). For the cryopreservation of the cells in monolayers, Watts and Grant reported that rat hepatocytes attached to collagen-coated dishes yielded fairly good recovery of functionally intact cells (11,18). However, for reasons that remain unclear, such methods have not yet achieved widespread recognition and application in the research community.

The collagen vitrigel membrane has several advantageous biological features. First, the vitrigel membrane has molecular permeability (15). A membrane permeation analysis reveals that low molecular mass materials such as glucose rapidly pass through the membrane, and even large molecules such as serum albumin follow the osmotic gradient and finally equilibrate within 1 week. This feature facilitates improved exposure of the cells to the culture medium and the cryopreservative, in contrast to cells on solid plastic plates, exposed only on the upper side. Another advantage of the collagen vitrigel membranes is their transparency, so that microscopy before and after preservation, and even a quantitative image analysis as reported herein, thus becomes feasible. In addition, cryopreservation using collagen vitrigel membranes is space saving. In the present study, we cryopreserved the collagen vitrigel membrane in 35-mm dishes but, because the membrane is a self-sustaining material, it could be stored in a thin plastic bag without other support.

The proliferation of the feeder cells for culture of ES cells is commonly prevented by mitomycin-C treatment, which changes their biological properties. Indeed, these cells could be well cultured only on UV-irradiated collagen vitrigel membranes, although untreated fibroblasts grew on such membranes whether or not they were irradiated. Similarly, their sensitivity to cryopreservation also differed. In our preliminary experiments, mouse fibroblasts could be stored frozen on plastic cell culture dishes without the collagen vitrigel membrane (data not shown). However, after mitomycin-C treatment, as routinely used for ES cell cultures, these cells did not survive cryopreservation on plastic culture dishes.

We conclude that the application of collagen vitrigel membranes and associated technologies is therefore expected to open up new avenues of investigation and increase the utility of cell cultures in biological research.