Electroporation buffer with cryprotective capabilities

Details Electroporation buffer with cryprotective capabilities Electroporation buffer with cryprotective capabilities

1999-10-05

2002-04-09

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Differentiated tissue or organ other than blood, per se, or...

Including freezing; composition therefor

C435S006120, C435S069100, C435S412000, C435S413000, C800S292000, C800S279000, C800S286000

Reexamination Certificate

active

06368784

ABSTRACT:

BACKGROUND OF THE INVENTION
Transfection of mammalian cells is a procedure whereby a molecule is placed into a cell by passing it through the cell's outer membrane without perturbing the viability of the cell. Molecules such as DNA, RNA, and proteins are often the transfected material.
Generally, the transfectant will not passively pass through the intact cell membrane. Its passage is aided by transient disruption of the membrane. Four methods are in common use.
Gene gun wherein the transfectant is first bound to a solid particle such as graphite or gold and shot at high velocity into the cell.
Liposome mediated transfection wherein a liposome complex is formed between lipid and transfectant. The liposomes interact with the cell membrane facilitating the passage of the transfectant into the cell.
Calcium phosphate, that chelates DNA and form a precipitate that is internalised.
Electroporation mediated transfection wherein an electrical field pulse is applied across the cell membrane which causes transient pore formation in the cell membrane. This enables the transfecting molecule to pass through the pore into the cell.
A prerequisite for all the above methods is that sufficient cells are present for transfection. This is achieved by prior expansion of cells by culture. The Gene gun is not usually a method of choice, as it is inefficient and requires that the cells are grown adhered to a surface. Liposome and electroporation mediated transfection are more efficient than the Gene gun. Liposome mediated transfection has been more popular than electroporation mediated transfection. This is partly due to the fact that liposome mediated transfection can be carried out on cells growing in solution and cells grown adhered to a surface. Electroporation mediated transfection is more efficient than liposome mediated transfection and is generally carried out on cells grown in solution. With the advent of electrophorus membranes (e.g., Corning) which allow electroporation of adherently grown cells, this imbalance is likely to be redressed. In both instances, standard cell culture media is a poor media for transfection. Therefore, culture media must be replaced with a suitable transfection solution prior to transfection.
The majority of cultured cells used in industry and academia are used for transient or stable (permanent integration of the transfected molecule) transfection studies. These cells lines are available through organizations such as the ETACC and ATCC. Cells lines are obtained as a frozen aliquot and clonally expanded by the purchaser. Once expanded, the cells are generally kept as a growing culture and as a frozen stock. This growing culture is the source of cells for transfection. Cells are generally frozen in a cocktail of enriched culture media and cryoprotectant. The most common cryoprotectant used is dimethyl sulfoxide (DMSO). However, glycerol, sucrose, trahalose and proline and others are sometimes used. Cryoprotectants are thought to aid cell survival by preventing ice nucleation in cells, which is a contributory factor to cell rupture upon freezing, while the culture media provides nutrients. The multiple factors involved in successful cryo-preservation of Animal cells have been extensively reviewed by Doyle et. al. 1988.
The generally used procedure for transfection is as follows:
(1) Culture cells to produce sufficient cells for transfection. This step can take several days.
(2) Transfect cells.
(3) Study transfected cells.
The disadvantages of this procedure is that pre-culture of cells for transfection requires an excessive amount of time and resources. Moreover, the requirement for transfection to be repeated each time an experiment is undertaken may lead to variability in transfection.
SUMMARY OF THE INVENTION
The instant invention is a cryoprotectant for electroporation ready cells comprising molecules that protect cells during the freeze thaw cycle prior to electroporation.
It is a high efficiency electroporation buffer containing antioxidants and other critical molecules that help in the regeneration process after electroporation.
The cryoprotectant/electroporation buffer comprises the cold protection of culture media with cryoprotectant and the transfection capabilities of electroporation media.
The dual purpose buffer combines a cell-freezing medium and a high efficiency mammalian cell transfection buffer.
The buffer is selected from a range of cryoprotectants as described in Stacey, 1997, Stange & Mitzner, 1996, Grout et al 1990 & Doyle et al 1988, that include DMSO, glycerol, sucrose, trehalose, and protein.
Preferred buffer is DMSO, glycerol or sucrose.
The buffer is useful simultaneously and directly as a high efficiency electroporation transfection buffer and as a buffer to freeze cells for long-term storage.
The invention is further a method of buffering transfected material comprising:
a) freezing the material in electroporation/cryoprotection media,
b) resuspending the material in water,
c) resuscitating by defrosting at room temperature for about 1 minute and then 37° C. for ~2 minutes, depending on cell type and apparatus available.
d) mixing an aliquot of material with cells and placing them in an electroporation cell followed by transfecting the material in a culture dish
In the invention, Cytomix, an intracellular ionic strength- and pH-mimicking buffer composed of 120 mM KCl; 0.15 mM CaCl
2
; 10 mM K
2
HPO
4
/KH
2
PO
4
, pH 7.6; 25 mM Hepes, pH 7.6; 2 mM EGTA, pH 7.6; and 5 mM MgCl
2
; pH adjusted with KOH, with added water and DMSO to final volume is resuspended and aliquoted into cryovials, wrapped and frozen to −20° C. over ~5 hours and then −70° C. overnight, deep freezing in nitrogen for ~5 days wherein the latter stage of freezing depends on the sophistication of the available apparatus and has an impact on the successful preservation.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention eliminates the need for repetitive pre-culture and therefore has the following advantages over the art. Experiments are initiated at the transfection step, significantly reducing the time and resources required greatly. The number of experimental steps is reduced and so is the likelihood of infection of cells. With all of the transfected cells from the same stock, there is a reduction of intra- and inter-experimental variation. This invention allows one to do a different experiment each day; one is not confined to one available batch.
In the instant invention, the need to pre-culture cells prior to transfection is eliminated by producing a frozen cell stock. This cell stock has the following characteristics: each aliquot has sufficient cells for a transfection; cells are viable when resuscitated; and cells are capable of immediate transfection upon resuscitation. The transfection technique used is already established, e.g., liposome transfection or electroporation. For each aliquot to have sufficient cells for a transfection, cells must first be cultured in sufficient quantities for stocks to be made. For the cells to be viable when resuscitated and be capable of immediate transfection upon resuscitation, a buffer which has a dual function is required. The buffer must function as a cryoprotectant and a transfection buffer. This buffer is the object of the instant invention.
The invention is a cryoprotectant/electroporation buffer useful simultaneously and directly as a high efficiency electroporation transfection buffer and as a buffer to freeze cells for long-term storage. This is achieved by combining the cold protection of culture media with cryoprotectant and the transfection capabilities of electroporation media. This alleviates the need for continuous cell culture and repeated transfections due to transfection variability of current methods.
Cytomix (Knight & Scrutton 1986) also sold as Optimix (marketed by EquiBio) is an intracellular ionic strength mimicking buffer. This buffer protects against osmotic rupture as it has the same osmotic strength as the cell contents. It also contains additional salts and other critical m

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