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Cotton rat lung cells for virus culture

A technology of viruses and cotton rats, applied in the field of generating organisms or pathogens, can solve the problems of high production costs and low titers

Inactive Publication Date: 2005-08-10
MERIAL LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the titer of PRRS virus on these cells is low, so the production cost is high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Obtaining Cotton Rat Lung (CRL) Cells

[0089] After excised from euthanized animals, lungs were placed in cell culture medium containing minimal essential medium F-15 (MEMF-15, Hyclone, catalog number SH30024.02) and 30 μg / ml gentamicin. The medium was supplemented with commercially available antibiotics (penicillin and streptomycin) and antimycotics (amphotercin B) at a concentration of 2% v / v. The lungs were digested at 37°C in a 50ml conical centrifuge tube containing 25ml of MEM F-15 containing 30μg / ml gentamicin, collagenase (Sigma, catalog number L-0130) and trypsin (Sigma , catalog number T-8003) at concentrations of 1 mg / ml and 1X strength (corresponding to a concentration of 2.5 mg / ml). After 30 minutes of digestion, fetal bovine serum (FBS) (Bio Whittaker) was added to a final concentration of 10% v / v. After the cells were blown off and clarified using a pipette, the supernatant containing the dissociated cells was collected and spr...

Embodiment 2

[0090] Embodiment 2: culture CRL cell

[0091] The residual FBS on the confluent (confluent) monolayer of CRL cells obtained in Example 1 was removed by rinsing with trypsin solution. 0.1% (1X) porcine trypsin and ethylenediaminetetraacetic acid (EDTA) (JRH Biosciences, cat. 2 3ml for flask, 150cm 2 Flasks were placed in a 37°C incubator covered with 5ml) of cells and monitored closely until cell detachment was complete. Blow off the cells using a pipette, and collect 3 ml of the cell suspension. At this point, harvested cells were cultured at a ratio of 1:4 with fresh cell culture medium containing MEM F-15, gentamicin at a concentration of 30 μg / ml, and FBS at a concentration of 10% v / v.

[0092] Keep CRL cells at 37°C, 5% CO 2 Grow in a partial pressure incubator. Confluent monolayers from a 1:4 split were confluent after about 4 days, while a 1:8 split produced confluent monolayers in about 7 days. This constitutes a passage.

[0093] Cells were t...

Embodiment 3

[0096] Example 3: Propagation of PRRS virus on CRL cells

[0097] Attenuated PRRS virus known to propagate on monkey kidney cells was used.

[0098] 75cm filled with 50ml medium 2 Propagation of PRRS virus was performed on a monolayer of CRL cells in flasks (T75 flasks) in a medium containing MEM F-15, gentamicin concentration 30 μg / ml and FBS v / v concentration 10%. Cells were incubated at 37°C for 24 hours until they were confluent before virus inoculation. After inoculating 1 ml of PRRS virus, the inoculated cells were incubated at 37° C. for 5-7 days. Viral growth was checked by indirect immunofluorescence antibody test (IFA) and supernatant titration. After freeze-thawing, the supernatant was harvested. This constitutes a crude culture of PRRS virus.

[0099] Indirect Immunofluorescence Antibody Test (IFA)

[0100] For IFA, cells were trypsinized, dispersed, collected and resuspended in fresh MEM F-15 medium containing 30 μg / ml gentamicin and supplemen...

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PUM

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Abstract

A cotton rat cell line, uses of cotton rat cells for growing, propagating, or culturing organisms, pathogens or viruses, such as PRRSV, and uses of the resultant organisms, pathogens or viruses, are disclosed.

Description

[0001] Related Applications / Introduced References [0002] This application claims priority to US Provisional Application No. 60 / 366,014, filed March 20, 2002, which is incorporated herein by reference. The foregoing application, all documents cited therein or in the course of its filing (“Application Cited Documents”) and all documents cited or referenced in the Application Cited Documents, and all documents cited or referenced herein (“herein cited Documents") and all documents cited or referenced in documents cited herein, and any manufacturer's instructions, descriptions, product inserts, and product listings for any product mentioned herein or in any document incorporated herein by reference are incorporated herein by reference , and can be used in the practice of the present invention. field of invention [0003] The present invention relates to novel cell lines and their uses, including methods for generating organisms or pathog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00A61K39/21A61K39/42C12N5/07C12N7/00C12N7/02
CPCA61K39/12C12N2770/10034A61K39/21A61K2039/5252C12N5/0688C12N7/00C12N2770/10051A61K2039/55566C12N2770/20051C12N5/0602C12N5/10C12N7/04
Inventor F·R·戴维M·E·塔纳S·K·雷迪
Owner MERIAL LTD
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