Anaerobic Growth and Maintenance of Mammalian Cell Lines

Summary

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Abstract

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO₂ in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2–3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H₂, CO₂, N₂) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage. Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Introduction

Cells from solid tumors, stem cell niches, and those lining the mucosal surfaces exist in environments that experience reduced oxygen levels, including anoxia^1,2,3,4. In normal physiologic states, oxygenation varies beyond that of hypoxia to anoxia (the complete absence of oxygen)^5,6.The realization that atmospheric oxygen adversely affects mammalian cell replication and that in vitro cell growth can be optimized under depleted oxygen conditions was reported in the earl....

Protocol

1. Prepare Media for Anaerobic Culture of Various Mammalian Cell Lines

1. Make the complete PS-74656 medium for an anoxic cell culture²⁵.
   1. Make the sterile nitrite stock solution (5 M; 100x) by dissolving 17.25 g of nitrite in 50 mL of distilled deionized water, then filter to sterilize it.
   2. Add 0.5 mL of the nitrite stock solution per 50 mL of low glucose DMEM (1 g/L of glucose) with 110 mg/L of L-glutamine, 584 mg/L of sodium pyruvate, and 10% fetal bovine serum (FBS; heat-inactivated).
      NOTE: The use of FBS concentrations greater than 10% are toxic. Of note, one cancer cell line tested ha....

Results

The strength of this protocol lies in its support of the longevity and the growth of multiple cell lines and in the recognition that there are a profound alteration and divergence in the cell morphology²⁵. The most critical element of this study is the transfer and maintenance of the cells under strict anaerobiosis. This requires an anaerobic chamber organized to maximize the protocol (Figure 1) and the assurance that the cells removed.......

Discussion

This method represents first-time mammalian cells that were cultured for extended periods of time in the absence of oxygen. Based on current observations, the anoxic growth capability via a non-fermentative pathway appears to be universal amongst mammalian cells lines²⁸, where the anaerobic growth resulted in phenotype divergence. This was observed for all cell lines tested. With the anaerobic cultivation, increasing proportions of the cells became rounded, developed a suspension-like pop.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Whitney A35 anaerobic chamber	Don Whitley Scientific	Microbiology International	The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals.
CO2 incubator	Fisher Scientific	3531
Tissue Culture Hood	Labconco	DO55735
pipet aid	Drummond	4-000-100
sterile transfer pipets	Santa Cruz	sc-358867
50 cc sterile conical centrifuge tubes	DOT	451-PG
vaccum jar	Nalgene 111410862889	BTA-Mall 5311-0250
DMEM high glucose (4.5 g/L)	CellGro	10-017-CM
DMEM low glucose (1 g/L)	CellGro	10-014-CV
FBS	VWR	1500-500
HBSS	VWR	20-021-CV
trypsin	VWR	25-052-CI
gentamicin	Gibco	15750-060
sterile pipets	CellTreat	229210B, 229205B
Tissue culture flask (T75 or T150)	Santa Cruz	sc-200263, sc-200264
24 well tissue culture treated dishes	DOT	667124
glad ziplock sandwich bags	Ziploc	Costco
inverted phase microscope (10, 20 40x objectives with camera mont)	Nikon Eclipse	TS100
trypan blue	Invitrogen	T10282
hemocytometer	Invitrogen	C10283
Countess Automated Cell Counter	Life Technologies	AMQAF1000
Rainin microtiter pipets	Mettler Toledo	Rainin Classic Pipette PR-100
microtiter tips	Santa Cruz Biotechnology	sc-213233 & sc-201717
test tube rack (50cc tubes)	The Lab Depot	HS29050A
sodium nitrite	Sigma	https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en®ion=US
clear boxes with lids	Rosti Mepal	Rubber maid
paper towels	In House
cell scraper	CellTreat	229310
PBS	In house prepared
70% Ethanol	Fisher Scientific	64-17-5
microcentrifuge tubes sterile	Santa Cruz	sc-200273
biohazard bags with holder (desktop)	Heathrow Scientific	HS10320
Nitrile Gloves	VWR	89428
oxygen electrode	eDAQ	EPO354
pH meter	Jenway	3510
pH paper/ pHydrion	Sigma Aldich	Z111813