In situ growth, freezing and testing of cultured cells

Details In situ growth, freezing and testing of cultured cells In situ growth, freezing and testing of cultured cells

2000-04-28

2002-10-29

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S001300, C435S004000, C435S005000, C435S007220, C435S374000, C435S395000, C435S404000, C424S093100

Reexamination Certificate

active

06472206

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides methods and compositions for the in situ growth, freezing and testing of cultured cells. In particular, the present invention provides methods and compositions for the long-term preservation of cells in ready-to-use formats for testing. In addition, the present invention provides rapid and easy to use means to diagnose viral and other infections. Furthermore, the present invention provides easy to use means to grow and store cells in situ for testing methods. Indeed, the present invention makes viral, chlamydial and other diagnostic methods accessible to small laboratories, including those without cell culture capabilities.
BACKGROUND OF THE INVENTION
As intracellular parasites (e.g., viruses and Chlamydia) require living cells in order to replicate, diagnosis of infection due to these organisms relies upon the use of either animals (e.g., suckling mice), embryonated eggs, or cell cultures. As cell cultures are much less expensive and are easier to work with than animals or embryonated eggs, cell cultures have long been the mainstay of diagnosis methods for intracellular parasites and viruses in particular. Indeed, cell cultures are the foundation upon which a virology laboratory is built. These cultures may be produced in house from animal tissues or organs, or more commonly, purchased from commercial suppliers.
Regardless of their sources, cell cultures must be maintained over time, in order to ensure a ready supply of cells for growth and diagnosis of infections caused by intracellular parasites. In the laboratory, mammalian cells are routinely frozen in order to minimize the opportunity for contamination of the cultures, guard against handling errors that could result in the loss of the culture, and minimize the number of cell lines that must be handled on a daily basis. Frozen cell culture stocks are also useful for minimizing genetic drift and shift, senescence, and undesirable phenotypic changes that can occur when continuous and finite cell lines are cultured for long time periods.
Freezing methods have been developed to minimize the impact of osmotic shock and intracellular ice crystal formation, two factors that contribute to the loss of cell viability during freezing. Cryoprotectants such as glycerol and dimethylsulfoxide (DMSO) are commonly used to help prevent cell death during freezing. In addition to the use of cryoprotectants, traditional methods use slow cooling (approximately 1° C. per minute) until the cells reach a temperature of −25° C. Once this temperature is attained, the cells can be rapidly cooled to −70° C. or −196° C. (i.e., liquid nitrogen temperature), without further loss of cell viability. Omitting the cryoprotectant or rapid freezing causes the formation of intracellular ice crystals which can rupture cell membranes and result in cell death. By slowly cooling the cells, the external medium becomes supercooled and ice crystal nuclei form in the extracellular fluid. This results in an extracellular environment that contains an artificially elevated salt gradient which, in turn, causes an osmotic gradient. This gradient causes water to diffuse out of the cells and the nonelectrolyte cryoprotectants to diffuse into the cells. This “dehydration” of the cells tends to minimize osmotic shock and intracellular ice crystal formation. (See e.g., Wiedbrauk and Johnston, “Mammalian Cell Culture Procedures, in Manual of
Clinical Virology
, pages 33-44 [Raven Press, New York, 1993], for a description of these events).
However, commonly used freezing methods require specialized equipment and training. In addition, hazardous chemicals such as DMSO are typically used. Furthermore, thawing of frozen cells maintained in liquid nitrogen poses risks such as explosion of the vials or tubes as well as the danger of loss of cell viability due to improper handling (e.g., slow, rather than rapid thawing). Once the cells have been thawed, the freezing medium must be removed and rinsed from the cells and the culture revived prior to use for growth and/or detection of intracellular parasites. Once revived, the cultures are often placed into formats suitable for the detection and identification of viruses, including multiwell plates (e.g., microtiter plates), tubes, and slides). Thus, the cultures must be transferred from their growth flask to these other formats prior to their use. This necessitates additional equipment and personnel time, prior to the use of the cultures as desired. What is needed are cell cultures and methods that are easy to use, readily available, particularly in ready-to-use formats, require little operator time and/or experience to use, and are reliable.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for the in situ growth, freezing and testing of cultured cells. In particular, the present invention provides methods and compositions for the long-term preservation of cells in ready-to-use formats for testing. In addition, the present invention provides rapid and easy to use means to diagnose viral and other infections. Furthermore, the present invention provides easy to use means to grow and store cells in situ for testing methods. Indeed, the present invention makes viral, chlamydial and other diagnostic methods accessible to small laboratories, including those without cell culture capabilities.
The present invention provides methods for the detection of intracellular parasites in a sample, comprising the steps of providing a cell culture comprising cells, wherein the cell culture has been frozen and thawed in situ on a substrate, and a sample suspected of containing at least one intracellular parasite; adding the sample to the cell culture to produce an inoculated culture; incubating the inoculated culture under conditions such that the intracellular parasite infects the cells of the cell culture to produce an infected culture; and observing the infected culture for the presence of the intracellular parasite within the cells of the cell culture. In some embodiments, the substrate is selected from the group consisting of glass and plastic. In some preferred methods, the glass is a glass coverslip. In still other preferred embodiments, the plastic substrate is the well of a multiwell plate. In some embodiments, the intracellular parasite is selected from the group consisting of viruses and bacteria.
In some embodiments, the observing comprises observing for the presence of cytopathic effect, while in other embodiments, the observing comprises observing for the presence of fluorescent cells. In still other embodiments, the observing comprises observing for the presence of blue cells. In some preferred embodiments, multiple observations are made. For example, in some embodiments, observing for cytopathic effect is combined with observing for fluorescent cells, and/or observing for blue cells. As described herein, in preferred embodiments, the observing for fluorescent cells is accomplished using labeled antibodies that recognize an antigen (e.g., viral or bacterial) present in the culture (i.e., due to the infection of the cells by the virus(es) and/or bacteria). However, it is not intended that the present invention be limited to any particular fluorescence product, substrate, enzyme, or color. Indeed, it is intended that multiple antibodies and fluorescence labels will find use in some embodiments of the present invention. For example, it is contemplated that the cells of the present invention will be tested using multiple antibody preparations with differing fluorescent labels. In addition, it is not intended that the present invention be limited to fluorescently labeled antibodies, as other detection means will find use with the present invention. Also as described herein, the observing for blue cells is associated with test methods such as those that involve the use of a reporter gene which indicates that a particular gene is being expressed. However, it is not intended that the present invention be limited to any particular gene, p

Affiliated with

Also associated with

Rating

In situ growth, freezing and testing of cultured cells does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with In situ growth, freezing and testing of cultured cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and In situ growth, freezing and testing of cultured cells will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2928160