Principles and practical issues for cryopreservation of nerve cells

doi:10.1016/j.brainresbull.2007.08.004

Brain Research Bulletin

Volume 75, Issue 1, 31 January 2008, Pages 1-14

https://doi.org/10.1016/j.brainresbull.2007.08.004 Get rights and content

Abstract

Nerve cells isolated from the brain have a number of research and clinical applications, not the least of which is their transplantation to patients with Parkinson's disease. Neural primary and precursor cells of several areas of the brain are potential candidates for transplantation and research. However, supply of suitable tissue is one of the major problems associated with the widespread application of such techniques. The ability to store such tissue for prolonged periods would greatly alleviate this problem. Cryopreservation allows indefinite storage, provided the storage temperature is sufficiently low. Whilst many of the potentially usable cell types have been shown to be capable of surviving cryopreservation to some degree, survival post-thaw needs to be considerably improved. Cryopreservation techniques applied to date are mostly crude and often adopted from those used for unrelated cell types. Studies involving cryopreservation of primary neural cells and stem cells are reviewed, the basic principles of cryopreservation explained and suggestions made for improvements to the low temperature storage of these cells.

Introduction

Cryopreservation allows the long term storage of cells, which has a number of applications. Clinically, cells may be stored by a patient for their own use at a later date. Cells may also be banked to facilitate donation to other patients. Having stored the cells, they can be distributed, making it easier to co-ordinate patient care, and avoiding the need to synchronise donor and recipient. The ability to store cells reduces wastage of cells if they cannot be used fresh, and thereby increases the supply of transplantable material. Storage also facilitates the pooling of cells from two or more donors where cells from a single donor are currently insufficient for a clinical effect. The ability to store cells allows for quality testing of a portion of the sample and allows time for microbiological and viral screening of the sample, and in some cases the donor; as well as preparation of the donor, where necessary. Cell storage is also valuable in scientific research; allowing archiving of material, repeated experiments from the same tissue source and, by allowing distribution of stored samples, facilitates research collaboration.

In terms of storage of cells, there are three options; culture at 37 °C, hibernation at 4 °C or cryopreservation. Culture at 37 °C is time consuming and can result in variation of the cell line with time, and hence is not a viable option. Storage at 4 °C has been reported to allow good survival of primary nerve tissue up to a maximum of 8 days [17], [18], [43]. Cryopreservation, on the other hand, can, if storage temperatures are maintained below −130 °C, allow virtually indefinite storage.

Cryopreservation is a routine procedure for the prolonged storage of many mammalian tissues such as blood, bone marrow cells, spermatozoa and embryos used clinically. For other cell types, cryopreservation has not been applied with sufficient success to enable it to be incorporated into routine clinical practice. Neural cells/tissue is one of these areas. In order to understand why one cryopreservation protocol is not sufficient to successfully preserve all cell types it is necessary to understand the events that occur during the freezing of cells.

Section snippets

Events during freezing

During storage at temperatures below −130 °C all biological events cease hence no deterioration of the sample occurs. What is potentially damaging to cells is cooling to, and warming from, the storage temperature. When cells are cooled to just below 0 °C, there will be no ice formation due to the presence of solutes. As the cells are cooled further, to between −5 and −15 °C, ice formation begins to occur. Ice forms preferentially in the extracellular medium. The formation of such extracellular ice

Cryopreservation of brain tissue

Interest in the cryopreservation of neural tissue followed the successful transplantation of such tissues, initially for studies of brain development and formation and regeneration of neuronal connections, and later for clinical transplantation. Disturbed brain function in animal models of neurodegenerative disorders has been ameliorated by neural transplantation [4]. Subsequently, transplantation of foetal primary neural tissue into the striatum of patients with advanced Parkinson's disease

Assessment of cryopreservation studies

Evaluation of studies involving cryopreservation is made difficult by the lack of detail given relating to the cryopreservation protocols used and the numerous variables involved, including tissue source; tissue preparation prior to freezing; treatment of tissue post-freezing; and the variety of ways in which investigators have quantified their results. Despite this there does, in general, appear to be agreement that good preservation of gross morphology and architecture of neural cells/tissues

Summary

There is obviously considerable room for improvement in cryopreservation techniques whatever the type of nerve cell to be preserved. Cryopreservation protocols applied to date result in a loss of cell yield or viability or in some cases both. In most cases cryopreservation has involved exposure to Me₂SO, slow-cooling, storage in liquid nitrogen, rapid warming and serial dilution of the cryoprotectant. Whilst such a technique has allowed a degree of survival post cryopreservation, recoveries

Conflict of interest statement

None.

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ReviewPrinciples and practical issues for cryopreservation of nerve cells

Abstract

Introduction

Section snippets

Events during freezing

Cryopreservation of brain tissue

Assessment of cryopreservation studies

Summary

Conflict of interest statement

Lancet Neurol.

Dev. Biol.

Brain Res.

Exp. Neurol.

Neurosci. Lett.

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Cryobiology

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Cell Transplant.

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

Res. Vet. Sci.

Brain Res.

Cryobiology

Brain Res.

Exp. Neurol.

Int. J. Dev. Neurosci.

Brain Res.

Brain Res.

Trends Neurosci.

Cryobiology

Lancet

Exp. Neurol.

Exp. Neurol.

J. Neurosci. Meth.

Brain Res.

Exp. Neurol.

Fertil. Steril.

Review
Principles and practical issues for cryopreservation of nerve cells