Preconditioned Cell Array Optimized for a Three-Dimensional Culture of Hepatocytes

Abstract

Three-dimensional culture procedures have attracted attention in various fields of cell biology. A newly developed cell array assisted in the formation of hepatocyte spheroids by two innovations: 1) micropatterning by a hydrophilic polymer, and 2) the use of bovine carotid artery-derived HH cells as feeder cells. The former contributes to the standardization of the spheroid size and the latter to the maintenance of the spheroids. We created a way to provide a ready-to-use cell array by cryopreservation of an HH feeder cell cultured array. After inoculation of HH cells on the cell array, the culture medium was replaced by freezing medium containing dimethyl sulfoxide. Thereafter, the array was frozen and stored in a −80°C deep freezer. At the start of the hepatocyte culture, the cryopreserved HH cell array was thawed by adding warmed (37°C) culture medium. The morphology and biological activities of the cryopreserved HH cells were intact, as confirmed by phase contrast microscopy and functional staining with calcein and formazan. The rat hepatocytes formed perfect spheroids on the cryopreserved HH cell array without any differences from those on the freshly prepared HH cell array. The CYP3A drug metabolism activities of the hepatocytes were well maintained on the cryopreserved and fresh cell arrays. The present protocol greatly shortened the time and labor required to prepare a cell array for culturing hepatocytes.

Introduction

The three-dimensional (3D) culture is a promising break-through in cell biology, tissue engineering, drug development, etc., because the 3D culture is more likely to reconstruct the in vivo behavior of cells than does the monolayer culture (2,4,8). Physiologically, most hepatocytes are at the static G₀ stage in vivo, but once isolated and cultured on the plate, the cells are put on growth signal (3). Concomitantly, the differentiated functions such as drug metabolism decrease in the conventional monolayer culture.

One unique 3D culture system cell array was developed to maintain hepatocytes in vitro by a combination of micropatterning of a polyethylene glycol-base hydrophilic molecule and the use of bovine carotid epithelial HH feeder cells (7). First, the micropatterning with a highly hydrophilic polymer standardizes the size of the hepatocyte spheroids at a 100-μm diameter at 100-μm intervals. The standardization of the spheroid size avoids inside necrosis and provides a uniform cell density in the culture well. Second, the feeder cells support the attachment and cultivation of hepatocytes (1). At present, a considerable number of commercial human hepatocyte lots are unable to be cultured because the hepatocytes readily lose their ability to attach to the culture matrix after cryopreservation. Human hepatocytes are indispensable to the drug development process, especially in regard to drug metabolism and pharmacokinetics (DMPK). Therefore, improved cell arrays are needed not only by cell biologists, but also by pharmaceutical researchers.

In this study, we developed a way to supply feeder cell-loaded arrays by cryopreservation. This technology greatly facilitates the use of the cell array by omitting the steps and time to prepare the feeder cells.

Materials and Methods

Chemicals, Cells, and Animals

Dulbecco's modified Eagle's medium (DMEM), Williams Medium E, and antibiotics (penicillin, streptomycin) were from GIBCO BRL, Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was from Gemini Bio-Products (West Sacramento, CA). Dimethyl sulfoxide (DMSO) (D2650), testosterone (UC339), and 6-β-hydroxytestosterone (UC282) were from Sigma Aldrich Fine Chemicals (St. Louis, MO). All other materials and chemicals not specified above were of the highest grade available.

Bovine carotid epithelial HH cells (JCRB0099) were obtained from the Japan Cell Research Bank. Male Sprague-Dawley rats (6–7 weeks old, specific pathogen free) weighing approximately 200 g were purchased from Sankyo Lab (Tokyo, Japan).

Preparation of Patterned Cell Array Disks

The procedures were performed basically according to a previously reported protocol (7). Glass disks (21 mm in diameter, 0.1 mm in thickness, Matsunami Glass, Osaka, Japan) were applied an aqueous solution of photosensitive hydrophilic polymers and the polymers were fixed by ultraviolet irradiation under patterned occlusion masks that have 100μm-diameter circles at 100-μm intervals. After washing of the unconjugated polymers, the cell array disks have a regular dot pattern where the cells can adhere. To culture cells, the cell array disks were placed in the wells of a 12-well plate.

Preparation, Cryopreservation, and Thawing of HH Cell Array

HH cells (3 × 10⁵) were inoculated onto the cell array and cultured in 1 ml of DMEM containing 10% FBS and antibiotics (100 U/ml penicillin, 100 U/ml streptomycin). After 24 h, the medium was replaced by 1 ml of ice-cold cryopreservation medium consisting of the cell culture medium and 10% DMSO. Immediately thereafter, the culture plates (HH cell arrays) were placed at −80°C in a deep freezer until use. The storage period examined in the present work was from 1 week to 3 months. In the transportation study, the cryopreserved HH cell arrays were packed in dry ice and carried by a conventional overnight courier service.

To thaw the cells, 1.5 ml of warmed (37°C) culture medium was added. After confirming that the last ice crystal had disappeared, the medium was changed to fresh culture medium. The survival of the cryopreserved feeder cells was observed by phase contrast and fluorescence microscopy. To culture the hepatocytes, isolated rat hepatocytes (3 × 10⁵ cells) were inoculated on a freshly prepared HH cell array, and the cryopreserved HH cell array 3 or 24 h after thawing. Rat hepatocyte isolation was performed by collagenase perfusion as described elsewhere (6). After adding rat hepatocytes, the cells were cultured in Williams Medium E containing 10% FBS, 1 μmol/L insulin, 1 μmol/L dexamethasone, and the antibiotics.

Functional Cell Viability Assay

The cells were stained with calcein and ethidium (Live/Dead Viability Cytotoxicity Kit, L3224, Molecular Probes, Eugene, OR, USA) to determine viability. The calcein-positive green cells were judged alive and the ethidium-positive red cells dead under fluorescence microscopy. The cells were also assayed calorimetrically for metabolic activity by formazan formation (Premix WST-1 Cell Proliferation Assay System, TaKaRa BIO, Shiga, Japan).

Assay for CYP3A Activity

The rat primary hepatocytes on the cell array were assayed for CYP3A activity by testosterone 6-β-hydroxylation as described previously (9). Briefly, the cells were incubated with 150 μmol/L testosterone in serum-free medium for 1 h. The harvested culture supernatant was analyzed for 6-β-hydroxytestosterone with HPLC using a reversed phase analytical column (TSKgel Super-ODS18197, TOSOH, Tokyo, Japan).

Results

Cryopreservation of the HH Feeder Cell Cultured Cell Array

As with the freshly prepared cell arrays (Fig. 1A, B), the cryopreserved HH cell arrays kept well-grown live HH cells on the disk with few dead or detached cells (Fig. 1C–F). There was no observable time course change in the cells 3 and 24 h after thawing (Fig. 1C, D and E, F, respectively). When the rat hepatocytes were inoculated onto the cryopreserved HH cell array 3 h after thawing, intact spheroids were formed at day 1 (Fig. 1G, H). The spheroids survived for 1 month or more, like those on a freshly prepared cell array. These results were reproducible with approximately 20 experiments including various conditions: storage periods varied from 1 week to 3 months, transportation between two collaborating facilities was performed. We have no evident trouble due to the present protocol of cryopreservation thus far.

Figure 1.

Morphological examination of the cryopreserved HH cell array. (A, C, E, G) Phase contrast micrograms and (B, D, F, H) fluorescent micrograms of calcein/ethidium staining (green cells are alive and red cells are dead). (A, B) Freshly prepared cell arrays 24 h after inoculation (control). (C, D and E, F) Cryopreserved HH cell arrays 3 and 24 h after thawing, respectively. The HH cell arrays were stored for 3 months at –80°C. There was no detectable difference regardless of cryopreservation. (G, H) Rat hepatocytes were inoculated on a cryopreserved HH cell array 3 h after thawing. Morphologically intact spheroids were observed (1 day after inoculation). Scale bar: 200 μm.

Functional Examination of Cryopreserved HH Cell Array

The metabolic activities of the cryopreserved HH cells and rat hepatocytes on the array are demonstrated in Figure 2A and B, respectively. Even after cryopreservation, the HH cells showed a sharp increase in formazan, a metabolite formed by mitochondrial dehydrogenase, at the same rate as with the freshly prepared HH cell array, thus indicating that there was no effect on the functional cell viability after freezing and thawing (Fig. 2A). Interestingly, the activities at 3 and 24 h after thawing were equal, suggesting that the HH cells started intracellular biological activities as soon as they were thawed.

Figure 2.

(A) Functional assessment of the cryopreserved HH cell array. Metabolic activity of the HH cells was estimated by the accumulation of formazan. The assay started 24 h after the inoculation of fresh HH cells (open circles), 2 h (open squares), and 24 h (closed squares) after thawing of the cryopreserved HH cell arrays (mean ± range, n = 2). The cryopreservation period was for 2 months. The rates of the accumulation of formazan were similar with all three conditions. (B) CYP3A activities of the rat hepatocytes on the freshly prepared and cryopreserved HH cell array were determined on day 1 to 4 by testosterone 6-β-hydroxylation. Rat primary hepatocytes were inoculated on fresh HH cells (open circles), 3 h (open squares), and 24 h (closed squares) after thawing of the cryopreserved HH cell array (mean ± range, n = 2). All three cultures were maintained under similar conditions.

The CYP3A activity of the hepatocytes on the cryopreserved cell array also confirmed the equal capability of feeding function (Fig. 2B). The hepatocytes on both the fresh and cryopreserved HH cell array showed stable activity of CYP3A, which is one marked feature of 3D culture. The initial activity of the isolated rat hepatocytes was 10.6 ± 0.8 nmol/well/min and, thus approximately 60–70% of the activity was maintained in the first 5 days of culture.

Discussion

Cryopreservation plays a fundamental role in the cell-associated technologies. Normally, cells, even adherent cells, are frozen as a suspension. With delicate cells such as primary hepatocytes, cryopreservation decreases the capability of cell attachment to a culture matrix and those cells are unable to be cultured further. We have proposed that in situ cryopreservation on vitrified collagen gel results in higher recovery in our other work (5). Using this concept, the present study applied this technology with a cell array system. Noteworthy points are: 1) the HH cell arrays can be frozen by a −80°C deep freezer, not by a controlled rate (program) freezer, and stored, as well, until use without the use of liquid nitrogen; 2) the cryopreserved HH cell arrays can be transported by a conventional overnight courier service; 3) the hepatocytes can be inoculated on the HH cell array directly after thawing, and the maintenance of the HH cell line and preculture on a cell array is unnecessary. These points are brought about mainly due to the cryoresistance of the HH cells and to the superiority of cryopreservation adherence in situ on the matrix (5).

Historically, the cell culture was developed with a purified cell population in a monolayer under artificial circumstances, but recent findings support the effectiveness of the 3D and mixed cell culture. These two methodologies facilitate the reconstruction of in vivo cell behavior. However, the increased number of experimental materials, especially biological materials like cells, complicates the laboratory work and quality control of experiments. Therefore, the ready-to-use cell array is expected to greatly help researchers. According to the original method, the HH cell arrays were prepared 1 day before the inoculation of the hepatocytes (7). In the present protocol, we confirmed that the 3-h preculture of the cryopreserved cell array was enough for the subsequent culture of the hepatocytes. Next, the inoculation of the hepatocytes just after thawing of the array will be attempted. Moreover, in combination with the improvement of cryoprotectants (6), the present method will be extended to other cell types, such as hepatocyte spheroids.