Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Summary

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

Abstract

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

Introduction

Spaceflight has been shown to impact many aspects of human physiology, including the immune system. Many studies have demonstrated evidence of immune dysregulation as a result of spaceflight in vivo and exposure to simulated microgravity (SMG) in vitro^1,2,3,4. One major aspect of the space environment that impacts human physiology is microgravity. Microgravity refers to the "weightlessness" experienced due to low gravitational forces in the space environment⁵. As humanity prepares fo....

Protocol

NOTE: The following steps should be completed inside a sterile biological safety cabinet.

1. Preparation of vessels for cell culture

1. Take the culture vessels out of the plastic packaging. Label each vessel on the rim according to what cell type/line is being used, whether it is the control (1G) or treatment (SMG), and any other relevant information.
2. Stabilize the vessel and open the fill port carefully, without touching the fill port cap peg/O-ring. Place the cap on an ethanol pad, with the peg/O-ring facing up, while the vessel is being filled. Do not remove the caps from the syringe ports.
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Results

This culturing method is considered successful if 1) the proliferation of the cells is approximately consistent across the control groups (and ideally all experimental groups), 2) the proliferation is appropriate given the seeding density, length of treatment, and the doubling time of the cell type/line, and 3) the viability of the harvested cells is 85% or higher (Table 1). Ideally, the resulting cells should be as healthy as they would be in standard cell culture, especially for use in subsequent exper.......

Discussion

As humanity prepares for longer space missions to the Moon and Mars, more research needs to be conducted to mitigate serious health risks in astronauts. One major aspect of the space environment that impacts human physiology is microgravity. Here, a cell culture method has been described for subjecting lymphocytes to SMG using a commercially available rotary cell culture system.

This protocol contains a few critical steps that may need to be optimized depending on the cell type or line that is.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Disposible High Aspect Ratio Vessel (HARV) (10 mL)	Synthecon	D-410	Gamma sterilized culture vessels (4/box)
Luer-Lok tip syringes (3 mL)	BD	309657	For attaching to the 10 mL HARVs
NK92 Cell-line	ATCC	CRL-2407
Rotary Cell Culture System (RCCS)	Synthecon	RCCS-4D	Rotating wall vessel device; 2D clinostat
Sarsedt 15 mL conical tubes	Fisher Scientific	50-809-220
Sarsedt 50 mL conical tubes	Fisher Scientific	50-809-218
Sarsedt sterile serological pipettes	Fisher Scientific	86.1254.001
T25 suspension culture flasks	Sarsedt	83.3910.502	For flask control