Expansion of mouse sertoli cells on microcarriers

Abstract

Background: Sertoli cells (SCs) have been described as the 'nurse cells' of the testis whose primary function is to provide essential growth factors and create an appropriate environment for development of other cells [for example, germinal and nerve stem cells (NSCs), used here]. However, the greatest challenge at present is that it is difficult to obtain sufficient SCs of normal physiological function for cell transplantation and biological medicine, largely due to traditional static culture parameter difficult to be monitored and scaled up.

Objective: Operational stirred culture conditions for in vitro expansion and differentiation of SCs need to be optimized for large-scale culture.

Materials and methods: In this study, the culturing process for primary SC expansion and maintaining lack of differentiation was optimized for the first time, by using microcarrier bead technology in spinner flask culture. Effects of various feeding/refreshing regimes, stirring speeds, seed inoculum levels of SCs, and concentrations of microcarrier used for expansion of mouse SCs were also explored. In addition, pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, and formation rates of lactic acid and ammonia, were investigated in culture.

Results: After 6 days, maximal cell densities achieved were 4.6 x 10(6) cells/ml for Cytodex-1 in DMEM/FBS compared to 4.8 x 10(5) cells/ml in static culture. Improved expansion was achieved using an inoculum of 1 x 10(5) cells/ml and microcarrier concentration of 3 mg/ml at stirring speed of 30 rpm. RESULTS indicated that medium replacement (50% changed everyday) resulted in supply of nutrients and removal of waste products inhibiting cell growth, that lead to maintenance of cultures in steady state for several days. These conditions favoured preservation of SCs in the undifferentiated state and significantly increased their physiological activity and trophic function, which were assessed by co-culturing with NSCs and immunostaining.

Conclusion: Data obtained in this study demonstrate the vast potential of this stirred culture system for efficient, reproducible and cost-effective expansion of SCs in vitro. The system has advantages over static culture, which has major obstacles such as lower cell density, is time-consuming and susceptible to contamination.

Figure 1

Microscopic observation of Sertoli cells (SCs) grown at different stirring speeds in spinner flask culture. SCs plated at 1 × 10⁵ cells/ml in 80 ml of DMEM in the presence of 3 mg Cytodex‐1 per ml at the stirring speed of 20, 30 and 40 rpm.

Figure 2

Effect of inoculum on viable cell density of Sertoli cells (SCs). Effect of inoculum on viable cell density for SCs grown in: (a) stirred microcarrier cultures and (b) control plates. Cells were plated at () 0.5 × 10⁵, (•) 1 × 10⁵ and () 2 × 10⁵ cells/ml in 80 or 2.5 ml of DMEM, for the stirred and control cultures, respectively. For stirred cultures, 3 mg of Cytodex‐1 per mL was used. □, ○, △ stand for viabilities (%) of SCs, plated at () 0.5 × 10⁵, (•) 1 × 10⁵ and () 2 × 10⁵ cells/ml in the figure. Media (50%) was changed every day from day 2.

Figure 3

Microscopic observation of Sertoli cells grown at different microcarrier concentrations. Sertoli cells (SCs) plated at 1 × 10⁵ cells/ml in 80 ml of DMEM with different microcarrier bead concentrations, observed on the 2nd day after inoculation.

Figure 4

Substrate concentration and metabolite profiles during culture of Sertoli cells (SCs) on Cytodex‐1 microcarrier beads. Concentration (, •, , ) and specific production/consumption (□, ○, △, ▽) rates of glucose (a), lactate (b), glutamine (c) and ammonia (d) in culture represented. Values displayed represent average of three independent experiments. Error bars indicate the standard deviation of duplicate cultures. SCs (1 × 10⁵ cells/ml) cultured with microcarrier bead concentration of 3 mg/ml. Media (50%) was changed every day, starting on day 2.

Figure 5

Optical and scanning electron micrographs of Sertoli cells (SCs) cultured on microcarrier beads under stirred culture conditions. SCs visualized using an optical microscope, day 2 (a), day 4 (b), day 6 (c) and day 8 (d) after MTT staining (200× amplification) for Cytodex‐1, respectively. SCs visualized using a scanning electron microscope, day 2 (e), day 4 (f), day 6 (g) and day 8 (h).

Figure 6

Identification of Sertoli cells (SCs) using immunocytochemistry with anti‐FAS‐L antibody. (a) SCs cultured on control plates. (b) Sertoli cells (SCs) cultured in stirred spinner flasks.

Figure 7

Photomicrograph of neuronal stem cells (NSCs) cultured alone and NSCs co‐cultured with Sertoli cells (SCs). (a) NSCs cultured in six‐well chamber slides. (b) NSCs co‐cultured with SCs obtained from two‐dimensional culture. (c) NSCs co‐cultured with SCs obtained from microcarrier bead suspension culture. All photographed on day 3 after inoculation (the scale bar is 5 μm). Neurite outgrowth was marked with.

Figure 8

Nerve stem cells (NSCs) immunocytofluorescently labelled with anti‐nestin antibody to identify neurospheres (day 6). (a) NSCs cultured on six‐well chamber slides. (b) NSCs cultured with Sertoli cells (SCs) obtained from two‐dimensional culture. (c) NSCs cultured with SCs obtained from microcarrier bead culture.