Chemical dissociation of cell aggregates

Abstract

The invention provides a novel method of dissociating anchorage independent and dependent cell aggregates. The invention also includes the cells resulting from the methods of the invention and the use of the cells in various applications requiring the generation of a single cell suspension.

Description

[0001] This application claims the benefit of priority under 35 USC§119(e) from United States Provisional application No. 60 / 553,947 filed on Mar. 18, 2004 which is incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention relates to a method for the chemical dissociation of cell aggregates producing minimal cell damage, maximizing cell viability and retaining the biological properties of the cells post dissociation. BACKGROUND OF THE INVENTION [0003] When cells are extracted from mammalian tissues and placed in culture, they tend to behave as either anchorage dependent or anchorage independent. Anchorage dependent mammalian cells grow by first attaching to a surface. These cells often divide until the surface on which they are attached is fully covered i.e. they divide and aggregate to form a confluent monolayer. Examples of anchorage dependent cells that are commonly used in industrial and research settings include Chinese Hamster Ovary cells (CHO cells)...

AI Technical Summary

Benefits of technology

[0018] The present invention provides a reproducible procedure for (i) the chemical dissociation of cell aggregates resulting in minimal damage to the cells, and (ii) the chemical dissociation of cells attached to surfaces resulting in minimal damage to the cells. The present invention, hereafter also referred to as chemical dissociation, is a significant advancement over currently used dissociation methods, allowing for increased viability, reduced cell damage, a significant increase in total number of cells generated and the maintenance of the functional properties of the cells upon subsequent sub-culture.

Problems solved by technology

However, recent evidence shows that the ECM is a complex and dynamic entity that can regulate the survival, development, migration, proliferation, shape and function of the cells in contact with it (Alberts et al., 2002).

Transplanting aggregates is undesirable because (i) aggregates can plug the delivery device (ii) it is difficult to estimate the number of actual cells that are delivered (iii) cells in aggregates are more susceptible to cell death due to the nutrient and oxygen mass transfer limitations that they suffer and (iv) aggregates are less likely to migrate to areas of damage, respond to local cues, and integrate into the host cellular architecture.

Cell sorting methods can only be used effectively on single cells.

Whereas all of these methods are effective in creating single cell suspensions, the excessive physical forces involved often result in a significant amount of cell death and cell damage.

In situations where the generation of a suspension of viable single cells is the ultimate goal, cell death and cell damage are extremely undesirable.

Mechanical dissociation can also result in the death of specific groups of important cells within a heterogeneous population.

Continually killing specific cell types during serial passaging could be detrimental to a cell line during long term culture.

In addition, the death of specific cell types could adversely impact results derived from procedures that rely on the generation of a single cell suspension such as flow activated cell sorting, and clonal and population analyses in the promising area of stem cell biology.

Moreover the manual nature of certain mechanical dissociation protocols (e.g. trituration, which is done by hand) often make it difficult compare measured values (such as cell viability) from different sources since dissociation efficiency varies between individuals.

However, not all cell types can be easily dissociated using enzymes.

For those cell types that are susceptible to enzymatic dissociation, it has been shown that enzymes can be detrimental to the cells and negatively impact the ability of the generated single cells to subsequently survive and / or divide.

For example, when neural stem cell (NSC) aggregates were dissociated using trypsin, the growth rate of the single cells in subsequent culture was found to have been adversely affected relative to single cells generated using mechanical dissociation (Sen, 2003).

In the extreme, enzymes can completely destroy cells.

However, due to the large quantities of extracellular matrix, mechanical dissociation of human neurosphere aggregates results in a much greater cell death relative to that caused during the mechanical dissociation of murine neural stem cell aggregates.

Currently, there are no methods available to expand these cells in vitro.

Large aggregates are undesirable since cells rapidly begin to die due to nutrient and oxygen limitations.

At present, there are no reliable or reproducible methods to accomplish this.

However, this method does not result in the generation of a single cell suspension.

Significantly increasing the intensity and duration of the mechanical dissociation process does not remove these aggregates, but rather, results in the death of large numbers of otherwise viable cells.

Thus, despite being the most commonly used procedure in this field, mechanical dissociation is not ideal.

It has recently been hypothesized that mutations to cells within the relatively quiescent stem cell compartment of mammary tissue results in the generation of breast cancer when rapid mitotic activity ensues.

One difficulty in conducting research with these cells is that they tend to aggregate when placed into serum-free culture.

In both cases there are issues related to generating a single cell suspension.

If the cells are attached as a monolayer, then mechanical dissociation is not effective, and enzymatic means (such as trypsin with EDTA) are routinely used to detach the cells, and subsequently break them into single cells.

However, enzymatic approaches are known to cause cell damage, or even death.

However, as described earlier, enzymes can be harmful to the cells.

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