Methods for preparing and using clonogenic fibroblasts and trans

Details Methods for preparing and using clonogenic fibroblasts and trans Methods for preparing and using clonogenic fibroblasts and trans

1996-08-22

2000-07-25

Priebe, Scott D.

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

435347, 435455, 435458, 435461, 424 932, A61K 3512, C12N 1563, C12N 1585, C12N 502

Patent

active

060933933

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a process for preparing clonogenic fibroblasts, to processes for genetically transfecting fibroblasts and to genetically transfected fibroblasts which are thus obtained.
In the therapy of a wide variety of diseases, it is desirable to supply particular biologically active molecules, which can also be produced by the human body, to the body in an increased, medicinally active dose. However, the medicinal supply of biologically active molecules which have been prepared outside the body also has the disadvantage that such active compounds have to be administered parenterally frequently and often even several times daily, with a single, subcutaneous administration often not being sufficient and intravenous doses which are administered several times daily being required instead.
SU 13 17 021 A1 relates to diploid stem cells from human skin and to embryonic muscle cells which have been isolated for the purpose of cultivating viruses.
SU 15 18 370 A1 relates to embryonic stem cell cultures of human skin and muscles for the purpose of producing diagnostic preparations.
Chapter 9 of the handbook written by R. Jan Freshney, "Culture of Animal Cells", 2nd Edition, Alan R. Liss Inc., New York, 1988, describes the isolation of tissue and primary (cell) cultures. On the following pages, a process for preparing human fibroblasts is described in which the tissue has been removed from the skin of donors within the context of a biopsy, whereupon the individual cells are isolated from the tissue, the cells which are present in the cell suspension are washed and the cells are converted into a tissue culture. However, this process comprises, exclusively within the context of isolating cells, a mechanical isolation of cells followed by an enzymic treatment with collagenase.
An object of the present invention is to provide a process by which cells of this nature are made available which have been altered in such a way that they are able to produce biologically active molecules.
The present invention discloses a process for preparing human clonogenic fibroblasts and also a process for genetically transfecting fibroblasts.


BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the mean doubling time of cell cultures prepared from various donors. The numbers behind the curves indicate the age of the respective donors.
FIGS. 2A-2F depict a comparison of proliferation of fibroblasts as a function of the manner in which the cells were prepared. The numbers in the lower light hand corners indicate the age of the donor.
FIGS. 3A (skin) and 3B (serosa) depict the proliferation of autologous fibroblasts from peritoneal cells.
FIGS. 4A and 4B depict the growth of diploid fibroblasts in the presence and absence of feeder cells.
FIGS. 5A, SE, and 5C depict the amount of TNF.alpha., GM-CSF and IL-6 detected in the supernatants of irradiated and non-irradiated cell cultures. D=days; H=hours.
FIG. 6 depicts the structure of the retroviral vectors which were used.
FIG. 7 depicts GM-CSF production (.eta.g/24 hr) by N2/CMV-GM-CSF/CMS 5#6 cells after irradiation with. 50 Gy. 10.sup.6 irradiated cells were plated on day 0 and the GM-CSF concentration in the 24 h culture supernatant was determined in a bioassay.
FIG. 8 depicts production of GM-CSF from irradiated populations on days 0, 5, and 7. The table gives the absolute value of the leucocyte subpopulations on treatment days 0, 5, and 7. Cyclo=cyclophosphamide, d0+d2 (i.p., 150 mg/kg). GM-CSF=cyclophosphamide, d0+d2 (i.p., 150 mg/kg)+rmGM-CSF, d3-d10 (s.c., 100 .eta.g, 2.times.daily). CMS5/GM-CSF=cyclophosphamide, d0+d2 (i.p., 150 mg/kg)+N2/CMV-GM-CSF/CMS5, d3 (s.c., 10.sup.7 cells).
FIG. 9 depicts the peripheral leucocyte counts in cyclosphosphamide-treated BALB/c mice with and without treatment with mGM-CSF or GM-CSF secreting fibrosarcoma cells.
FIGS. 10A, 10B, 10C, 10C, and 10D depict the leucocyte count as numbers of cells per .eta.l as a function of time after inoculation with non-irradiated CMS-5 cells transfected with pCMV.GCSF iresNEO (FIGS. 10A and 10C) or p

REFERENCES:
patent: 4423145 (1983-12-01), Stampfer et al.
patent: 4804627 (1989-02-01), Hammerling et al.
Crystal, R.G. et al, (1995), Science 270: 404-410.
Panis et al. (1992) C.R. Acad. Sci. Paris 315: 541-544.
Fireshney, R.I. (1988) `A Manual of Basic Technique` 2nd Edition, Alan R. Liss, Inc. New York pp. 107-126.
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