Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of storing cells in a viable state
Reexamination Certificate
2000-04-07
2002-06-25
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of storing cells in a viable state
C435S372300, C435S372000, C435S004000, C435S006120, C435S007240, C435S007210
Reexamination Certificate
active
06410321
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
The present invention relates to methods for lyophilizing eukaryotic cells and isolating intact nucleic acids from such cells, and kits for using the same.
BACKGROUND
Methods of lyophilizing or “freeze-drying” viruses, biologically active molecules, and bacteria are routinely used in laboratories. Typically, the cells or molecules to be lyophilized are suspended in a solution that allows recovery of the desired activity after the freezing process. Ideal formulations for lyophilization solutions should provide a stabilizing environment for a finite time before lyophilization; provide good thermal and freezing properties during lyophilization; and cryoprotect the desired activity.
Previous methods of lyophilization have focused primarily on using sugars in preservation of proteins in eukaryotic cells and preservation of bacteria. For example, the primary ingredient of solutions for lyophilizing bacterial cells is a sugar at a concentration of 5-10%, used to stabilize the cells (Greaves,
Fundamental Aspects of Freeze
-
drying Bacterial and Living Cells,
in
Aspects Theoriques et Industriels de la Lyophilisation,
407-410 (Rey ed. 1964). However, effective eukaryotic cell lyophilization methods for preserving nucleic acids, particularly RNA, have not been established. The inclusion of sugar such as lactose in an isotonic eukaryotic cell lyophilization formulation did not give high yields of nucleic acid isolated from lyophilized cells. The inclusion of preserving materials, such as low concentrations of methanol or ethanol in the lyophilization solutions was also found to be ineffective for isolation of intact nucleic acid after lyophilization.
Methods of long term preservation of nucleic acids from eukaryotic cells are desirable due to the use of molecular biological approaches for disease diagnosis and forensics. In such assays, positive and negative controls from a reference source are particularly useful. Clinical samples from previous patients and cell lines with known genotypes are often used as sources of experimental control cells. However, clinical samples from patients are typically available in limited amounts. Cultured cell lines are convenient sources of control cells, however, the use of freshly cultured cells for each assay is problematic because of the time, cost, and labor needed for growth and maintenance of cultured cells.
SUMMARY OF THE INVENTION
The present invention therefore provides an isotonic solution that is nuclease free and is used as a lyophilization solution for eukaryotic cells, allowing isolation of intact DNA and RNA from the cells. This invention is useful for long term storage and preservation of nucleic acid from eukaryotic cells. The invention also provides standard nucleic acid samples for use as controls in assays such as diagnostic and forensic assays.
In one aspect the invention provides a method for long term preservation of nucleic acid contained within eukaryotic cells, the method comprising the steps of: (1) lyophilizing an aqueous solution of intact eukaryotic cells where said solution is isotonic to the cells and is nuclease free; and (2) maintaining the lyophilized cells under sealed conditions sufficient to avoid contact with atmospheric humidity.
In one embodiment, the aqueous solution has been treated with diethyl pyrocarbonate. In another embodiment, the aqueous solution comprises sodium chloride at a concentration of between 0.8 and 1.% by weight to volume. In yet another embodiment, the aqueous solution comprises a standard phosphate buffered saline solution. In a further embodiment, the pH of the solution is between 6.8 and 8.2.
In one embodiment, the eukaryotic cells are mammalian. In another embodiment, the cells are human. In yet another embodiment the cells are lymphocytes. In a further embodiment, the cells are infected with an RNA virus. In a further embodiment, the cells are cultured prior to lyophilization.
In another aspect, the invention provides a method for isolating intact nucleic acid from lyophilized cell comprising the steps of: (1) lyophilizing an aqueous solution of living eukaryotic cells where said solution is isotonic to the cells and is nuclease free; (2) maintaining the lyophilized cells under sealed conditions for at least 30 days; (3) unsealing the cells; (4) denaturing the cellular proteins to create a mixture of intact nucleic acid and denatured cellular proteins; and (5) isolating intact nucleic acid from the mixture of denatured cellular proteins and nucleic acid with the proviso that the cells are not revived.
In one embodiment, the isolated nucleic acid is ribonucleic acid. In another embodiment, the cells are lymphocytes.
In another aspect the invention provides a collection of standardized, sealed vials containing lyophilized eukaryotic cells for use as controls in diagnostic assays wherein the cells after 4 weeks at −20° C. have more than 50% of their 18S rRNA intact as measured by gel electrophoresis.
In one embodiment, the cells are lymphocytes. In another embodiment, the cells are infected with an RNA virus. In a further embodiment, the vial contains an inhibitor of RNase in an amount effective to reduce degradation of RNA.
In another aspect the invention provides a nucleic acid hybridization kit comprising lyophilized eukaryotic cells.
In one embodiment, the cells are human. In another embodiment, the cells are lymphocytes. In yet another embodiment, the cells are infected with an RNA virus.
In another embodiment, the kit further comprises cell free nucleic acid selected to hybridize to a known target nucleic acid. In yet another embodiment, the kit further comprises a labelled nucleic acid. In a further embodiment, the nucleic acid hybridization assay uses RNA extracted from the lyophilized cells.
In another embodiment, the nucleic acid hybridization assay is an amplification based assay. In yet another embodiment, the amplification based assay is a ligase chain reaction based assay or a polymerase chain reaction based assay. In a further embodiment, the kit further comprises at least one PCR primer pair. In another embodiment the kit further comprises reverse transcriptase. In yet another embodiment the RNA is transcribed into complementary DNA during the assay.
In another embodiment, the kit further comprises a vial containing the cells wherein the vial is sealed to prevent atmospheric humidity from contacting the cells.
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Chemical Abstracts 122(13):158688c, 1995.*
Chomczynski, Piotr (1993) “A Reagent for the Single-Step Simultaneous Isolation of RNA, DNA and Proteins from Cell Tissue Samples”,BioTechniques15(3):532-535.
Gill, Santokh S., et al. (1996) “Ensuring Revovery of Intact RNA from Rat Pancreas”,Molecular Biotechnology, 6:359-362.
Jennings, Thomas, A. (1997) “Effects of formulation on lyophilization, part 1”,IVD Technology3(2):42-49.
Jennings, Thomas, A. (1997) “Effects of formulation on lyophilization, part 2”,IVD Technology3(2) :42-49.
Greaves, R.I.N (1964) “Fundamental aspects of freeze-drying bacteria and living cells”,Aspects Théoriques et Industriels de la lyophililsation, 407-410.
Cossman Jeffrey
French Cynthia
Lin Ching-I Patsy
Wallace Robert Bruce
Bio-Rad Laboratories, Inc.
Prats Francisco
Townsend and Townsend / and Crew LLP
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