CA2107559A1 - Immortalization of endothelial cells - Google Patents
Immortalization of endothelial cellsInfo
- Publication number
- CA2107559A1 CA2107559A1 CA002107559A CA2107559A CA2107559A1 CA 2107559 A1 CA2107559 A1 CA 2107559A1 CA 002107559 A CA002107559 A CA 002107559A CA 2107559 A CA2107559 A CA 2107559A CA 2107559 A1 CA2107559 A1 CA 2107559A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
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- Genetics & Genomics (AREA)
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- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Vascular Medicine (AREA)
- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates, in general, to endothelial cells.
In particular, the present invention relates to a microvascular endothelial cell (or a cell line) obtained from human skin and immortalized and a method to establish such a line.
In particular, the present invention relates to a microvascular endothelial cell (or a cell line) obtained from human skin and immortalized and a method to establish such a line.
Description
Wo92/17~6s PCT/US92/02499 21û7~
IMMORTALIZATION OF
ENDOTHELIAL CF~TS
BAC~GROUND OF THE INVENTION
Field of the Invention The present invention relates, in general, to endothelial cells. In particular, the present invention rPlates to immortalized microvascular endoth~lial cells obtained from human skin.
Backaround Information In the last 10 years, the accumulation of information about the endothelium has led to the realization that this is a tissue which is not just a target for injury, but by undergoing alterations in functions, metabolism and structure, it directly influences the evolution and outcome of vascular injury, inflammation and immune reactions like graft rejection and tumor metastasis (Cotran, R. (1987) Am. J. Pathol. 129, 3: 407-413). Factor VIII-related antigen is an endothelial cell product involved in the aggregation of platelets;
megakaryocytes are the only other cell type known to express this antigen (Cotran, R. (1987) Am. J.
Pathol. 129, 3: 407-413). Upon activation by molecules like Interleukin-1 and tumor necrosis factor, these cells up-regulate their expression of leukocyte specific adhesion molecules like ICAM-l and ELAM-1 (Pober, J.S. (1988) Am. J. Pathol. 133, 3: 426-433; Butcher, E.C. et al. (1986) J. Cell.
Biochem. 30, 2: 121-131); interferon-gamma is associated with the expression of Class II major histocompatibility antigens (Dvorak, H.F. et al.
~U~;ST~TUTE SHEE7 ,- . . . - - . . - . . .. . .
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WO92/17~69 PCT/US92/0~ ~
IMMORTALIZATION OF
ENDOTHELIAL CF~TS
BAC~GROUND OF THE INVENTION
Field of the Invention The present invention relates, in general, to endothelial cells. In particular, the present invention rPlates to immortalized microvascular endoth~lial cells obtained from human skin.
Backaround Information In the last 10 years, the accumulation of information about the endothelium has led to the realization that this is a tissue which is not just a target for injury, but by undergoing alterations in functions, metabolism and structure, it directly influences the evolution and outcome of vascular injury, inflammation and immune reactions like graft rejection and tumor metastasis (Cotran, R. (1987) Am. J. Pathol. 129, 3: 407-413). Factor VIII-related antigen is an endothelial cell product involved in the aggregation of platelets;
megakaryocytes are the only other cell type known to express this antigen (Cotran, R. (1987) Am. J.
Pathol. 129, 3: 407-413). Upon activation by molecules like Interleukin-1 and tumor necrosis factor, these cells up-regulate their expression of leukocyte specific adhesion molecules like ICAM-l and ELAM-1 (Pober, J.S. (1988) Am. J. Pathol. 133, 3: 426-433; Butcher, E.C. et al. (1986) J. Cell.
Biochem. 30, 2: 121-131); interferon-gamma is associated with the expression of Class II major histocompatibility antigens (Dvorak, H.F. et al.
~U~;ST~TUTE SHEE7 ,- . . . - - . . - . . .. . .
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WO92/17~69 PCT/US92/0~ ~
2~07~9 (1986) Hum. Pathol. 17, 2: 122-127).
Morphologically, endothelial cells can express facultative traits of tissues from which they were derived, including Weibel-Palade bodies and cobblestone growth pattern (Cotran, R. (1987) Am. J.
Pathol. 129, 3: 407-413; Xarasek, M.A. (1989) J.
Invest. Dermatol. 93: 335-385).
Primary human microvascular endothelial cells have a limltod llfe span of about ~-lC
passages and have specific growth requirements.
Early methods of tissue culture required tho US2 sf high concentrations of serum, Sarcoma 180 conditioned medium and multiple growth factors for optimal growth. Human serum requirements can be decreased or substituted with fetal bovine serum by incorporating 2% pre-partum maternal serum in the medium (Karasek, M.A. (1989) J. Invest. Dermatol.
93: 335-385). These cells can also be stimulated by the addition of agents such as cholera toxin, dibutyryl cAMP and isobutyl methyl xanthine which activate adenyl cyclase prolonging the growth rate and morphology (Iuder, R.M. et al. ~1990) J. Cell Physiol. 142: 272-283). In the absence of cAMP, cultured vascular endothelial cells undergo pronounced changes in their morphology and functioDal properties; cells turn from epithelial to spindle shape and lose some of their ability to express HLA-DR antigens in response to interferon-gamma (Tuder, R.M. et al. (1990) J. Cell Physiol.
142: 272-283).
Various normal and neoplastic, as well as differentiated embryonic cells of human origin, have been transformed and immortalized by intact SV40 . . , ~ . . . ..
,,;
SUBSTlTUTc SHEET
~ . ' ' . ,' ' 21U7 j~9 virus including human umbilical cord endothelial cells (SacX, G.H., Jr. (1981) In Vitro 17, 1: 1-19;
Gimbrone, ~.A. ~ Jr. et al. (1976) Cell 9: 685-693).
Papova viruses constitute one of the simplest group of DNA tumor viruses and have been the most studied (Aaronson, S.A. (1970) J. Virol. 6, 4: 470-475).
The SV40 transfected human endothelial cells did not exhibi~ ~actor VIII related antigen expression nor show char~st~rlstic Weibel-~alade bodies (Gi~brone, îO i~.~., ~.. ~t al. (1976) Cell 9: 685-693). Human ~m~i lic21 vel n endothelial cells have also been immo' talized by exposure to murine sarcoma viruses containing the l'v-ras~ or "v-mos" oncogenes (Faller, D.V. et al. (1988) J. Cell Physiol. 134: 47-56).
These cells expressed Factor VIII related antigen and contained Weibel-Palade bodies. An endothelial cell line derived from mouse lymph node stroma which ; retains most functional characteristics of normal mouse endothelial cells has been described (O~Connell, X.A., and Edidin, M. (l990) J. Immunol.
144, 2: 521-525); transient infection was performed ~ using whole virus SV40 strain 4A to immortalize ; these cells.
The cells according to this invention exhibit a number of utilities. For example, the cells can be used to study the immediate adherence of HDMEC to graft vascular surfaces, for example:
angioplasty and endarterectomy. The cells can also be used in pre-coating vascular grafts (with endothelial cells).
i The cells can be used in metabolic studies of lipid and lipoprotein metabolism, arachidonic acid metabolism, hemostasis factors, and endothelial !
~'' ' ' ' ' - . '.' : ' ~ , `
,. - .
- . - .- . -. .
W092/1~569 PCT/US92/0~ ~
21~7~
derived vasoactive substances such as endothelin (ET). The cells can also be used in studies of angiogenesis, wound healing, leukocyte adherence and adhesion molecule expression (intracellular expression as well). Further, the cells can also be used in genetic studies aimed at the isolation o~
endothelial cell specific gene regulatory products and creation of cDNA libraries for endothelial cell specific g2nes.
One sXilled in the art will apprecia t2 that the cells of th2 invention can be used in pharmacologic s-udies as substrates for the screening of various agents as inhibitors of inflammation or modulators of cell adhesion molecule expression or in the cosmetic industry for toxicity testing. The cells, if tumorigenic, can be used in studies of endothelial cell tumor formation and potentially useful in specific problems, such as Kaposi sarcoma (or the transforming effects of chemicals or other agents on diploid human cells).
The cells can also be used for viral or parasitic growth or detection. The cells can be used to produce products (for example: cytokines or lymphokines) which may be secreted into the medium or isolated from the cell surface.
SUMMARY OF THE INVENTION
It is a general object of this invention to provide a microvascular endothelial cell.
It is a specific object of this invention to provide a primate immortalized microvascular endothelial cell.
SJ~ 5TUTE SHEE ~ :
. . . . . . . . . ., .; .......... .. . ..... .. - ....... .
.
W092/~7S69 PCT/US92/02499 2~7~ ~j9 It is a further object of this invention to provide a primate immortalized microvascular endothelial cell line.
I~ is another object of this invention to S provide a method of establishing a cell line of primat~ o~t~ li z~d microvascular endothelial cells.
~ u~th~r objects and advantages of the ~r~s_~ n~ -n ~ e clQar .rom the d~scription that -~-ollo-~s.
~ -. one Pm~odiment, the present invention relatQs to 2 microvascular endothelial cell (or cell line~ ~her2in the c~11 (or cell line) is obtained from a primate skin and is immortalized.
In another embodiment, the present invention relates to a method of establishing a cell line of immortalized microvascular endothelial cells derived from primate skin comprising:
(1) introducing DNA which encodes SV40 large T
antigen into the cells and (2) c~lturing the cell line.
ERIEF DESCRIPTIO~ OF THE DRAWINGS
Figure 1. (A) Phase-contrast microscopy of a pure culture of human dermal microvascular endothelial cells (HDMEC) showing typical "cobblestone" morphology. (B) Phase-contrast microscopy of a culture of SV 4OT-transformed human dermal microvascular endothelial cells. They also have a "cob~lestone" appearance and are indistinguishable from non-transformed human dermal microvascular endothelial cells.
SUBSTITUT~ S.
' ,, ., . ' , . " .: " . ." ~ ' . ' ' . . ~ ' , . . ' ' ' '~. ' . "
WO92/1756s PCT/US92/0~99 2 !~ '3 7 .~ ~ 9 6 Figure 2. (A) Direct immunofluorescence - -microscopy of human dermal microvascular endothelial cells using acetylated LDL. The cells show granular cytoplasmic staining indicating uptake of the acetylated LDL, typical of endothelial cells. (8) Direct immunofluorescence microscopy of CDC/EU.XMEC-1. The staining is typical of endothelial cells and indistinguishable from that shown in Figure 2A.
~igure 3. (~) Phase-contrast microscopy of human dermal microvascular endothelial cells gro~n on matrigel. Long, tu~ular, c~llular ~xtensions are seen at eight hours of cultur~. This is typical of the morphologic differentiation that endothelial cells undergo when cultured on this matrix. (B) Phase-contrast microscopy of CDC/EU.HMEC-l cells cultured on matrigel.
; Morphologic differentiation is noted that is very similar to that seen in Figure 3A. --Figure 4. CDC/EU.HMEC-1 (Transformed ~ -HDMEC) cells were grown in HDMEC medium supplemented with different concentrations of human serum. They ~-were seeded at 1.7 x 10 cells/flask, cultured for eight days at 37C, 5% C0" har~ested, and counted. -~
Cells grew at all concentrations of human serum that were tested but did so in a concentration-dependent fashion. (Untransformed human dermal microvascular endothelial cells will not grow below 20% normal human serum.) Figure 5. CDC/EU.HMEC-1 or untransformed human dermal microvascular endothelial cells were seeded at 5 x 10' cells/25 cm' flask and grown for 10 days at 37C 5% CO, with various concentrations of human serum. Cells were harvested every 24 hours SUBSTITUTE SHEET ~ .
. .. .
j' : ' ''. ' ' .' ' ,, , . .. : . . ' . ', '. ' . , ! : .
WO 92/17569 PCT/US92/0~99 21~7rj59 for 10 days and counted. (Open boxes = CDC/EU.HMEC-1, 30Q0 52~ ; solid boxes = C~C/EU.HMEC-l, 1% serum;
open diamonds = CDC/EU.HMEC-1, 0% serum; solid diamonds = .~M~C, 307 serum.) CDC/EU.HMEC-1 cells grow best with 30~ human serum supplementation, but will surv v~ at 0% human serum.
Figure 6. HLA-DR Expression on CDC-HMEC-1. Cell-i ~2r_ untr2ated or treat~Ad with Interferon-gamm2 -^?- 5~tan days. E~prassion of HLA-DR W2S
rerA^~ e~ ACSc-^~n analysis using PE-conjugated anti-h~an ~LA-DR a~tisar~m.
Morphologically, endothelial cells can express facultative traits of tissues from which they were derived, including Weibel-Palade bodies and cobblestone growth pattern (Cotran, R. (1987) Am. J.
Pathol. 129, 3: 407-413; Xarasek, M.A. (1989) J.
Invest. Dermatol. 93: 335-385).
Primary human microvascular endothelial cells have a limltod llfe span of about ~-lC
passages and have specific growth requirements.
Early methods of tissue culture required tho US2 sf high concentrations of serum, Sarcoma 180 conditioned medium and multiple growth factors for optimal growth. Human serum requirements can be decreased or substituted with fetal bovine serum by incorporating 2% pre-partum maternal serum in the medium (Karasek, M.A. (1989) J. Invest. Dermatol.
93: 335-385). These cells can also be stimulated by the addition of agents such as cholera toxin, dibutyryl cAMP and isobutyl methyl xanthine which activate adenyl cyclase prolonging the growth rate and morphology (Iuder, R.M. et al. ~1990) J. Cell Physiol. 142: 272-283). In the absence of cAMP, cultured vascular endothelial cells undergo pronounced changes in their morphology and functioDal properties; cells turn from epithelial to spindle shape and lose some of their ability to express HLA-DR antigens in response to interferon-gamma (Tuder, R.M. et al. (1990) J. Cell Physiol.
142: 272-283).
Various normal and neoplastic, as well as differentiated embryonic cells of human origin, have been transformed and immortalized by intact SV40 . . , ~ . . . ..
,,;
SUBSTlTUTc SHEET
~ . ' ' . ,' ' 21U7 j~9 virus including human umbilical cord endothelial cells (SacX, G.H., Jr. (1981) In Vitro 17, 1: 1-19;
Gimbrone, ~.A. ~ Jr. et al. (1976) Cell 9: 685-693).
Papova viruses constitute one of the simplest group of DNA tumor viruses and have been the most studied (Aaronson, S.A. (1970) J. Virol. 6, 4: 470-475).
The SV40 transfected human endothelial cells did not exhibi~ ~actor VIII related antigen expression nor show char~st~rlstic Weibel-~alade bodies (Gi~brone, îO i~.~., ~.. ~t al. (1976) Cell 9: 685-693). Human ~m~i lic21 vel n endothelial cells have also been immo' talized by exposure to murine sarcoma viruses containing the l'v-ras~ or "v-mos" oncogenes (Faller, D.V. et al. (1988) J. Cell Physiol. 134: 47-56).
These cells expressed Factor VIII related antigen and contained Weibel-Palade bodies. An endothelial cell line derived from mouse lymph node stroma which ; retains most functional characteristics of normal mouse endothelial cells has been described (O~Connell, X.A., and Edidin, M. (l990) J. Immunol.
144, 2: 521-525); transient infection was performed ~ using whole virus SV40 strain 4A to immortalize ; these cells.
The cells according to this invention exhibit a number of utilities. For example, the cells can be used to study the immediate adherence of HDMEC to graft vascular surfaces, for example:
angioplasty and endarterectomy. The cells can also be used in pre-coating vascular grafts (with endothelial cells).
i The cells can be used in metabolic studies of lipid and lipoprotein metabolism, arachidonic acid metabolism, hemostasis factors, and endothelial !
~'' ' ' ' ' - . '.' : ' ~ , `
,. - .
- . - .- . -. .
W092/1~569 PCT/US92/0~ ~
21~7~
derived vasoactive substances such as endothelin (ET). The cells can also be used in studies of angiogenesis, wound healing, leukocyte adherence and adhesion molecule expression (intracellular expression as well). Further, the cells can also be used in genetic studies aimed at the isolation o~
endothelial cell specific gene regulatory products and creation of cDNA libraries for endothelial cell specific g2nes.
One sXilled in the art will apprecia t2 that the cells of th2 invention can be used in pharmacologic s-udies as substrates for the screening of various agents as inhibitors of inflammation or modulators of cell adhesion molecule expression or in the cosmetic industry for toxicity testing. The cells, if tumorigenic, can be used in studies of endothelial cell tumor formation and potentially useful in specific problems, such as Kaposi sarcoma (or the transforming effects of chemicals or other agents on diploid human cells).
The cells can also be used for viral or parasitic growth or detection. The cells can be used to produce products (for example: cytokines or lymphokines) which may be secreted into the medium or isolated from the cell surface.
SUMMARY OF THE INVENTION
It is a general object of this invention to provide a microvascular endothelial cell.
It is a specific object of this invention to provide a primate immortalized microvascular endothelial cell.
SJ~ 5TUTE SHEE ~ :
. . . . . . . . . ., .; .......... .. . ..... .. - ....... .
.
W092/~7S69 PCT/US92/02499 2~7~ ~j9 It is a further object of this invention to provide a primate immortalized microvascular endothelial cell line.
I~ is another object of this invention to S provide a method of establishing a cell line of primat~ o~t~ li z~d microvascular endothelial cells.
~ u~th~r objects and advantages of the ~r~s_~ n~ -n ~ e clQar .rom the d~scription that -~-ollo-~s.
~ -. one Pm~odiment, the present invention relatQs to 2 microvascular endothelial cell (or cell line~ ~her2in the c~11 (or cell line) is obtained from a primate skin and is immortalized.
In another embodiment, the present invention relates to a method of establishing a cell line of immortalized microvascular endothelial cells derived from primate skin comprising:
(1) introducing DNA which encodes SV40 large T
antigen into the cells and (2) c~lturing the cell line.
ERIEF DESCRIPTIO~ OF THE DRAWINGS
Figure 1. (A) Phase-contrast microscopy of a pure culture of human dermal microvascular endothelial cells (HDMEC) showing typical "cobblestone" morphology. (B) Phase-contrast microscopy of a culture of SV 4OT-transformed human dermal microvascular endothelial cells. They also have a "cob~lestone" appearance and are indistinguishable from non-transformed human dermal microvascular endothelial cells.
SUBSTITUT~ S.
' ,, ., . ' , . " .: " . ." ~ ' . ' ' . . ~ ' , . . ' ' ' '~. ' . "
WO92/1756s PCT/US92/0~99 2 !~ '3 7 .~ ~ 9 6 Figure 2. (A) Direct immunofluorescence - -microscopy of human dermal microvascular endothelial cells using acetylated LDL. The cells show granular cytoplasmic staining indicating uptake of the acetylated LDL, typical of endothelial cells. (8) Direct immunofluorescence microscopy of CDC/EU.XMEC-1. The staining is typical of endothelial cells and indistinguishable from that shown in Figure 2A.
~igure 3. (~) Phase-contrast microscopy of human dermal microvascular endothelial cells gro~n on matrigel. Long, tu~ular, c~llular ~xtensions are seen at eight hours of cultur~. This is typical of the morphologic differentiation that endothelial cells undergo when cultured on this matrix. (B) Phase-contrast microscopy of CDC/EU.HMEC-l cells cultured on matrigel.
; Morphologic differentiation is noted that is very similar to that seen in Figure 3A. --Figure 4. CDC/EU.HMEC-1 (Transformed ~ -HDMEC) cells were grown in HDMEC medium supplemented with different concentrations of human serum. They ~-were seeded at 1.7 x 10 cells/flask, cultured for eight days at 37C, 5% C0" har~ested, and counted. -~
Cells grew at all concentrations of human serum that were tested but did so in a concentration-dependent fashion. (Untransformed human dermal microvascular endothelial cells will not grow below 20% normal human serum.) Figure 5. CDC/EU.HMEC-1 or untransformed human dermal microvascular endothelial cells were seeded at 5 x 10' cells/25 cm' flask and grown for 10 days at 37C 5% CO, with various concentrations of human serum. Cells were harvested every 24 hours SUBSTITUTE SHEET ~ .
. .. .
j' : ' ''. ' ' .' ' ,, , . .. : . . ' . ', '. ' . , ! : .
WO 92/17569 PCT/US92/0~99 21~7rj59 for 10 days and counted. (Open boxes = CDC/EU.HMEC-1, 30Q0 52~ ; solid boxes = C~C/EU.HMEC-l, 1% serum;
open diamonds = CDC/EU.HMEC-1, 0% serum; solid diamonds = .~M~C, 307 serum.) CDC/EU.HMEC-1 cells grow best with 30~ human serum supplementation, but will surv v~ at 0% human serum.
Figure 6. HLA-DR Expression on CDC-HMEC-1. Cell-i ~2r_ untr2ated or treat~Ad with Interferon-gamm2 -^?- 5~tan days. E~prassion of HLA-DR W2S
rerA^~ e~ ACSc-^~n analysis using PE-conjugated anti-h~an ~LA-DR a~tisar~m.
3~T~ ~ T~n DESCRIPTION OP THE INVENTION
The present invention relates to human microvascular endothelial cells.
In one embodiment, the present invention relates to microvascular endothelial cells obtained from primate skin (preferably, human skin or foreskin) and immortalized. In a prefeEred embodiment, immortalization is effected by introducing DNA encoding SV-40 large T antigen into the skin-derived endothelial cells. A preferred cell line comprising such immortalized cell is designated CDC/EU.HMEC-l. This cell line has been dQposited in accordance with the Budapest Troaty with the American Type Culture Collection, 12301 ParXlawn Drive, Rockville, MD 20852, USA as ATCC
D-signation CRL 10636. The deposit was received January 8, 1991 and was accepted by this International Depository Author ty.
The deposited human microvascular endothelial cell line from human foreskin was immortalized by .
~U''S7-lTlJTE SHEET
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WO92/17569 PCT/US92/0~9g ~ ~ ~7559 transfecting freshly isolated cells with an eukar~otic voctor containing the early region of the SV40 virus large T antigen. These cells (CDC/~U.'IM~C~ orm microtubules on matrigel, they take up acetylated low density lipoprotein, express Factor VIII-related antigen and express HLA-DR
antigen upon exposure to gamma-interferon; all of which a ~ characteristics of normal microvascular endothelial ce1ls. ~dditionally, these cells shaL2 0 'ho s ~ 1~= epi~hPlioid co~blestone growth pattern of normal microvascular ,ondothelial cells.
Di.f2ren. - om normàl HD~EC, this new cell line reauires low or no concentration(s) of human or fetal bovine serum for growth, has a shorter lS doubling time than HDMEC, do not require epidermal growth factor or hydrocortisone for growth and do not require gelatin or fibronectin-coated surfaces $or attachment. Control cells which were not transfected and did not contain SV-40T DNA died off after two additional passages. Immortalized cells are thus defined as cells which remain alive after ten passages following introduction of DNA.
Ideally, they replicate or divide indefinitely, maintain morphologic and physiologic characteristics of the tissue of origin, and grow at lower serum reguirements and increased cell density. In particular, the DNA encodes the SV-40 large T
antigen and can be introduced via transfection.
In a further embodiment, the present invention relates to a method of establishing a cell line of immortalized microvascular endothelial cells derived from primate skin (preferably human skin or foreskin). The method comprises introducing DNA
SUBSTITUTE SHEET
:: ' ~ , ,,, ', . . :' ' W092~t7569 PCT/US92/02499 21~7~
g encoding the S~40 large T antigen into the cells under condit~ons such that the microvascular endothelial cells ~ecome immortalized and culturing the c2 11 Th~ pres~nt invention is described in further detail in the following non-limiting examol~s.
~ ~T~T ~;
~h_ -.olio~ing orotocols and experimental details are re-rerenced in the examples that follow.
~ .
endothelial cells (HDMEC!. HDMEC have been isolated from human foreskins by the following technigue.
Foreskins are cut into 3 mm squares and placed in phosphate-buffered saline (PBS) containing 0.3%
trypsin (Sigma Chemical Co., St. Louis, M0) and 1%
ethylenediamine tetracetic acid (Sigma) at 37C for 10 minutes. The skin segments are washed with HBSS
several times and placed in a petri dish in HBSS
with the keratinized surfaced down. They are then individually compressed with the side of a scalpel blade to express microvascular fragments from the cut surfaces of the skin. The microvascular segments in 1 ml of HBSS are layered onto a 35%
solution of Percoll (Pharmacia AB; Uppsala, Sweden) in H9SS that has been previously spun at 30,000 g for 10 minutes at 4C to create a gradient. The gradient is then spun at 400 g for 15 minutes at room temperature. The fraction with a density less than 1.048 g per ml, which is rich in microvascular fragments, is removed. Those portions of the SUBSTITUTE SHEET
. . . - - . -- .
. . .
.. . ., - -: ... : . . - ,.. . ~ .
, - . ~ ,' ' ~ ' ' WO92/17569 PCT/VSg2/0~99 21~75'a9 gradient containing the microvascular segments are applied to a human fibronectin (Advanced Biot~chnologies; Silver Spring, MD) precoated area 10 mm in diame.Pr in the center of a 60-mm tissue-culture dish. The dishes are then incubated at 37Cin a moist incubator in 5% C02 overnight. Unattached cells are removed by washing with HBSS. The attached c~lls ar~ th2n viewed with an inverted phase-contrast microscope in 2 ~iologic hood, and nor.e.~c~h~'ial cells a~e removed by detaching them with ~ ~5-gaus2 sterile needle. The growth medium for _h~s2 c21' S consists of 200 ml Endothelial Basal M2di~m ~CDB 131 (Clonetics, San Diego, CA), 75 ml - human serum (Irvine Scientific, Santa Ana, CA), Dibutyryl CAMP O.5 mM (Sigma Chemical Co., St. Louis, M0; Tuder et al. (1990) J. Cell.
Physiol 142:272-283), Antibiotic-antimycotic ~olution 1% (final concentration) (Gibco, Grand Island, NY), hydrocortisone 2 ~m (final concentration) and epidermal cell growth factor 5 ng/ml (final concentration). Transfected cells were grown in the same medium without CAMP. The resulting cell cultures are consistently 100% pure a~ assessed by morphological and immunochemical criteria. The normal cells can be continuously pa~saged up to 10 times.
Vec~. The vector used in the transfection is dQsignated as SV-40T. It has the sequence that codes for the transformation protein of SV-40 large T antigen and RSV-LTR cloned into the Eco Rl site of the PBR322 plasmid.
~:lleSTlTUTE SHEET
.. . : . .: . , ' ,. . '~ ', ' .. : - -. .
W092~17569 PCT/US92/0~99 2 ~
Transfection. Primary human foreskin endothelial cells were ln their 6th passage from isolation when transfected. The procedure followed has been described ~y Graham and van der EB (Virology, 52:456-467, 1973). Some minor modifications were introduc~d to this ~rocedure. All surfaces to which endothelial cells attach were pre-coated with a solution of 0.1~ gelating in 0.01~ phosphate buffer saline ?h 7 a (73S) . ~he colls were plated onto 6-well plates a, 3.5 x lCs celia/-~ell and incuba-ced @
37C and 5~ C0. overnight ~e_ore transfection. The amount or vector DNA ~sed ~as ~ ~g per well.
Following ihe transfection ~rocedure, the plates were incubated overnight ~ 37C with 5% C0" then the lS contents of each well transferred to individual 25 cm2 plastic flasks with 0.2 ~ filter cap (Costar).
- Some of the flasks contained dibutyryl cAMP 0.5mM
(final concentration). Flasks were split 1:4 as they became confluent.
.
Detection of SV-40T viral antiqen. To detect expression of the SV-40T in the immortalized endothelial cells (C~C/EU.HMEC-1) an enzyme-linked immunoassay was performed. Cell line SV-T2 a Balb/3T3 mouse embryo line infected with SV-40 (ATCC
# CCL 163.1) was used as a positive control, a peripheral lymphocyte lysate was used as a negative control. Cell-free lysates were obtained by adding 0.5 ml of 0.5% deoxycholate.HCl in O.OlM P8S pH 8.2 and 100 ~l of a 1 mM solution of phenylmethyl sulfuflouride in 95% ethanol to 10 million cells.
This mixture was incubated Q 4C for 30 minutes and then centrifuged @ 4000 RPM for 15 minutes @ 4C.
.
. :
'. SU2STlT~iTE SHEET
.
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.. . ~ . . - - -. . .... ... ....
WO92/17569 PCT/US92/0~99 21Q7~9 The supernatant which possibly contained the SV-40 large T antigen was removed and saved.
For the ELISA test, the antigens were attached by suctioning onto nitrocellulose paper using a ~inifold II Slot Blotter (Schleifher &
Schuell, ~eD~e, NH). After the attachment procedure, the sheets were incubated for 24 hr with 2% s'clm mll'i; ~ 0.01 ~ PRS Q 4C to prevent nonspDcific i~inding. Afterwards, the skim mil~
0 solu- o.. is ~~-o-ta zr,d the ni.-ocellulose washed three tlmDs--iith 0.1~ TwePn-20 in PBS Q RT . ~ouse moncc'^r.21 a..__~o~ to .he S'v-40 large T viral antisen was added a~ a dilution of 1:1000 in PBS/Tween and incubated for 30 min @ 37C on an orbital shaker. The monoclonal antibody to SV-40T
antigen was obtained from the supernatant of the mouse hybridoma cell line Pab 101 (ATCC # TIB 117).
The nitrocellulose sheets were then washed three --times with Tween/PBS and an additional wash @ 37C
on the orbital shaker for 15 min. Goat anti-mouse - horseradish peroxidase labelled monoclonal Ab (Bio~ad, Ric~mond, CA) was added at a 1:2000 dilution and allowed to incubate for 30 minutes Q
37C shaking. Later, the nitrocellulose sheet was washed three times with Tween/PBS. A
diaminobenzidine (Sigma Chemical Co., St. Louis, MO) (DAB) solution consisting of 25 mg of DA~, 50 ml of PBS, and 20 ~l of 30i% HtO, was added and color allowed to develop. The nitrocellulose sheet was rinsed with deionized water to stop the enzymatic reaction.
, ~' ~E~ t ~UTE Si JE~
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W092/17~69 PCTtUS92/0~99 2t~7~ '9 Expression of SV-40T antiaen. Cell-free lysates of CDC/EU.HMECI and SV-T2 (positive control) cells expressed the SV-40T viral antigen by ELISA and Western Blot. Negative cont~ol (hu~an peripheral blood lymphocyte lysate) cells were negative.
Characterization of endothelial cell culturPs.
Representative cultures of ~D;IEC and SV 4OT .iDMEC
(CDC/EU.HMEC-1) are charact2rized in thre~ ways.
Cultures are evaluate~ by nve-.ad has--con'_2~' microsccpy to d2tormine whethor 'h~ c_lls have he characteristic cob~laston2 .~or?hology e~^ ~ndotheliai cells. Cells are fixed in 90~ methanol at -20C for 10 minutes, washed and stained with a 1:40 dilution of rabbit anti-human factor VIII (Atlantic Antibodies; Scarborough, ME) for 30 minutes followed by FITC conjugated goat anti-rabbit IgG. They are washed three times and then viewed under a fluorescent microscope. Cells, unfixed, are incubated with acetylated low-density lipoprotein (10 ~g/ml), labeled with 1,l'dioctadecyl-1,3,3,3'3'-tetramethyl indocarbocyanine perchlorate (Dil-AC-LDL) (Biomedical Technology, Inc.; Stoughton, MA) at 37C in medium 199 without growth supplement or fetal calf serum for 4 hours. Dil-Ac-LDL is a biologic probe incorporated by living endothelial cells and, to a lesser extent, by monocytes or macrophages. The media is then removed, and the cells are washed twice and visualized in a fluorescence microscope with standard rhodamine excitation emission filters. Evaluation by all three techniques reveals that pure cultures of both types of endothelial cells are routinely attained.
SU~STITUTE SHEET
,. . - .:
W092tl7569 PCT/US92/0~ ~
2 ~ 5 9 Diffe Qr;tla.ion OL endotheli~l cells. Matrigel, an extrac. o h~ ~-IS sarcoma that contains basement membran~ co-~or.ents (Collaborative Research;
Bedford, MA), is applied to 24- or 96-well cell-cul.ur~ pla.2s as eit.~e a ch-cX or thin film and then incubat_d at 37C. This temperature induces selli~g ~ e ~t act. ~:DMEC or CDC/~U.HMEC-1 are th~r ~la~ . 3 '~ m_=_ igQ~ e cells at.ach rapid ~, ~ilG -~ n i-^~ ~OU_ S elongated process2s lo are 9bs~ d, and æ-^ter 8 hours the endothelial cell cul.u;^~s snow a~undant nP works of branching and anastomosing co~ds or cells. Bv light microscopy, most of the cords show a central translucent structure along their long axis, which suggests the presence of a lumen. By 8 hours, the endothelial cells form an interconnected network of anastomosing cells that by low-power light microscopy have a "honeycomb" appearance. These endothelial cells express factor VIII-related antigen before, during, and after tube formation. They are also metabolically active, since they ta~e up acetylated low-density lipoprotein. Transmission electron microscopy of cells cultured on matrigel for 18 hours demonstrates that cross-sections of the tube-like structures contain a lumen surrounded by cells.The membranes of the cells forming the lumen of the tubes connect with one another by interdigitating cytoplasmic processes.
Flow cytometry. Analysis of cell-surface molecules on HDMEC and CDC/EU.HMEC-1 was performed using direct immunofluorescence and flow cytometry.
SUBSTITUTE SHEET
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,. - . . . - . . ~ . , ~ .. . .
- , - . :,. . :- : : ,; , : :
... . . ..
WO92/17569 PCT/US92~0~ ~
2~7~ 9 Cytometric analysis was performed on a FACscan Flow Cytometer (3ecton-Dickinson, I~c.; ~ountain View, CA). This instrument provides data regarding cell number, forward angle light scatter, side scatter, and red and green fluorescence. Approximately 10,000 cells per test sample were analyzed in these studies. HDMEC and CDC/EU.HMEC-1 to be analyzed in these studies will be removed from tissue-culturo flas~s with 6-10 ml of 5~M Er~.~ and '~ 3S.~ to a~ d any loss of trypsin or dispase-sensi~ e enao~heliai cell epitopes. After incu~ating ror 30 minutes at 37C, an equal volume or HBSS with C~T~ and Mg+- is added to inactivate the EDTA, and the cells are washed three times. The cells are separated into aliquots of 10~ cells/tube, pelleted, supernatant discarded, and 20 ~1 of undiluted monoclonal antibody is added. The cells are lightly vortexed and incubated for 30 minutes on ice. The cells are washed three times and then either stained with an appropriate second-step antibody or ana-lyzed directly in one-step staining procedures.
Phenotv~ic Characterization of SV 40T HDMEC
The cell line, CDC/EU.HMEC-l is in its 40th passage. Control cells which did not contain SV-40T DNA or cAMP growth supplement after tran~fection died off after two additional passages after transfection. CDC/EU.HMEC-1 assumed a "cobblestone" morphology when cultured on gelatin-coated tissue culture dishes when cultured incomplete HDMEC media. Their morphology was essentially indistinguishable from HDMEC (Figure 1).
SU.~, T i ~ UTE SHcFT
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21075a9 It was noted, however, that when allowed to become hyperconLluent, the CDC/EU.HMEC-l were capable of growing to a higher density and that the cells appeared nat~.al;y smalier t~an H3~EC under these conditions.
C3C~-TJ.~EC-' ar.à HDYEC both s.ained positively fo- Factor VIII wh_n examined by direct immunofluorDscer.~ cth yres 3_ c~lls also demons.-a~ ?-''~ c. aa_;ila.2d iow aensi -y lipopro~ein a^~ no ~s e~osur~ (~isure 2).
In or-er .o det-r~ine whe-her CDC/Eu.X~EC-1 were c2pabl ~ o~^ mor~h^logic di.ferentiation into tubes, th~se cDlls w~re cultured on m2trigol. It was previously shown that HDMEC will form capillary-like structures when cultured on the basement - ~ -membrane-line matrix (Xubota, Y., et al. (1988) J.
Cell Biol. 167:1589). CDC/EU.HMEC-l demonstrated tube formation after 8 hours of culture on matrigel which was indistinguishable from that of HDMEC
(Figure 3).
P~oliferation Studies It was previously demonstrated that optimal growth of HDMEC requires specialized growth medium supplemented with 30% human serum (Kubota, Y., et al. (1988) J. Cell Biol. 107:1589 and personal observation). Decreasing human serum to 20% or below essentially halts proliferation. In order to determine human serum requirements for CDC/EU.HMEC-l, comparison growth studies were performed with routine HDMEC medium supplemented with 30%, 20%, 10%, 5%, or 1% human serum. The results showed that although the cells grew best at ~UBSTIT'i .~
.. .. . . . .
- . : . ; . . . .
2 - .
. .
.: - :. - ` ' : .
- ' , . .
, . , ' . .. ' - ` . . ..
WO92~l7s69 PCT/US92/0 2 1 ~ 7 rj ~ 9 30% human serum, there was growth even at a concentration of 1% human serum (Figure 4).
Population doubling time ranged from 53.6 hours for cells cultured in media containing 30~ human serum to 85.6 hours for cells cultured in media containing 1% human serum. Further studi~os ex2mining srs~th curves of CDC/EU.HMEC-1 showed that CDC/EU.~IEC-1 were capable of replicating in routine HDM~C cul~uro media devoid of serum (Figure 5 ).
Cell Surface Molecule Ex~rsssion Previous studies have shown that HDMEC s~r~ss a number of cell adhesion molecules on their surface including ICAM-1, LFA-3, CD44, and Class I, but lack 15 constitutive expression of Class II molecules (Fleck, R. et al. (1986) J. Invest. Dermatol.
86:475). HDMEC and CDC/EU.HMEC-1 were compared for expression of these cell surface antigens. Both normal and transfected cells expressed ICAM-l, LFA-20 3, CD44, and Class I, but did not express Class II
(Table 1). However, stimulation of HDM~C and CDC/EU.HMEC-1 with IFN-gamma (500 ~/ml, 72 hrs) resulted in induction of Class II cell surface antigen expression (Figure 6). The CDC/EU.HMEC-1 25 expressed more than 3 times as much CD44 and more than twice as much ICAM-1 and LFA-3 than did nontransfected HDMEC. Class I expression was roughly equivalent on both types of cells.
T~3LE I. Comparison of expression of selected immunologically relevant cell-surface antigens on untransformed and CDC/EU.HMEC-... . . . . . . . . . . .
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W092tl7569 PCT/US92/0~99 2.~ ~7~5~
1 human dermal microvascular endothelial c211s.
XLi'~EC COC~,;U. ~IEC-l IC~-l + +
L~A- 3 _ , CD4 4 +
Class II - - -Class TT~
~a.'e. stimu'a ion Fo~ n ~.~Jlth 500 u~ml o,^ l~-ga~ma.
* * * * *
All publications mentioned hereinabove are hereby incorporated in their entirety by reference.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.
S!J~,TUTE SKEEl r . ~
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The present invention relates to human microvascular endothelial cells.
In one embodiment, the present invention relates to microvascular endothelial cells obtained from primate skin (preferably, human skin or foreskin) and immortalized. In a prefeEred embodiment, immortalization is effected by introducing DNA encoding SV-40 large T antigen into the skin-derived endothelial cells. A preferred cell line comprising such immortalized cell is designated CDC/EU.HMEC-l. This cell line has been dQposited in accordance with the Budapest Troaty with the American Type Culture Collection, 12301 ParXlawn Drive, Rockville, MD 20852, USA as ATCC
D-signation CRL 10636. The deposit was received January 8, 1991 and was accepted by this International Depository Author ty.
The deposited human microvascular endothelial cell line from human foreskin was immortalized by .
~U''S7-lTlJTE SHEET
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WO92/17569 PCT/US92/0~9g ~ ~ ~7559 transfecting freshly isolated cells with an eukar~otic voctor containing the early region of the SV40 virus large T antigen. These cells (CDC/~U.'IM~C~ orm microtubules on matrigel, they take up acetylated low density lipoprotein, express Factor VIII-related antigen and express HLA-DR
antigen upon exposure to gamma-interferon; all of which a ~ characteristics of normal microvascular endothelial ce1ls. ~dditionally, these cells shaL2 0 'ho s ~ 1~= epi~hPlioid co~blestone growth pattern of normal microvascular ,ondothelial cells.
Di.f2ren. - om normàl HD~EC, this new cell line reauires low or no concentration(s) of human or fetal bovine serum for growth, has a shorter lS doubling time than HDMEC, do not require epidermal growth factor or hydrocortisone for growth and do not require gelatin or fibronectin-coated surfaces $or attachment. Control cells which were not transfected and did not contain SV-40T DNA died off after two additional passages. Immortalized cells are thus defined as cells which remain alive after ten passages following introduction of DNA.
Ideally, they replicate or divide indefinitely, maintain morphologic and physiologic characteristics of the tissue of origin, and grow at lower serum reguirements and increased cell density. In particular, the DNA encodes the SV-40 large T
antigen and can be introduced via transfection.
In a further embodiment, the present invention relates to a method of establishing a cell line of immortalized microvascular endothelial cells derived from primate skin (preferably human skin or foreskin). The method comprises introducing DNA
SUBSTITUTE SHEET
:: ' ~ , ,,, ', . . :' ' W092~t7569 PCT/US92/02499 21~7~
g encoding the S~40 large T antigen into the cells under condit~ons such that the microvascular endothelial cells ~ecome immortalized and culturing the c2 11 Th~ pres~nt invention is described in further detail in the following non-limiting examol~s.
~ ~T~T ~;
~h_ -.olio~ing orotocols and experimental details are re-rerenced in the examples that follow.
~ .
endothelial cells (HDMEC!. HDMEC have been isolated from human foreskins by the following technigue.
Foreskins are cut into 3 mm squares and placed in phosphate-buffered saline (PBS) containing 0.3%
trypsin (Sigma Chemical Co., St. Louis, M0) and 1%
ethylenediamine tetracetic acid (Sigma) at 37C for 10 minutes. The skin segments are washed with HBSS
several times and placed in a petri dish in HBSS
with the keratinized surfaced down. They are then individually compressed with the side of a scalpel blade to express microvascular fragments from the cut surfaces of the skin. The microvascular segments in 1 ml of HBSS are layered onto a 35%
solution of Percoll (Pharmacia AB; Uppsala, Sweden) in H9SS that has been previously spun at 30,000 g for 10 minutes at 4C to create a gradient. The gradient is then spun at 400 g for 15 minutes at room temperature. The fraction with a density less than 1.048 g per ml, which is rich in microvascular fragments, is removed. Those portions of the SUBSTITUTE SHEET
. . . - - . -- .
. . .
.. . ., - -: ... : . . - ,.. . ~ .
, - . ~ ,' ' ~ ' ' WO92/17569 PCT/VSg2/0~99 21~75'a9 gradient containing the microvascular segments are applied to a human fibronectin (Advanced Biot~chnologies; Silver Spring, MD) precoated area 10 mm in diame.Pr in the center of a 60-mm tissue-culture dish. The dishes are then incubated at 37Cin a moist incubator in 5% C02 overnight. Unattached cells are removed by washing with HBSS. The attached c~lls ar~ th2n viewed with an inverted phase-contrast microscope in 2 ~iologic hood, and nor.e.~c~h~'ial cells a~e removed by detaching them with ~ ~5-gaus2 sterile needle. The growth medium for _h~s2 c21' S consists of 200 ml Endothelial Basal M2di~m ~CDB 131 (Clonetics, San Diego, CA), 75 ml - human serum (Irvine Scientific, Santa Ana, CA), Dibutyryl CAMP O.5 mM (Sigma Chemical Co., St. Louis, M0; Tuder et al. (1990) J. Cell.
Physiol 142:272-283), Antibiotic-antimycotic ~olution 1% (final concentration) (Gibco, Grand Island, NY), hydrocortisone 2 ~m (final concentration) and epidermal cell growth factor 5 ng/ml (final concentration). Transfected cells were grown in the same medium without CAMP. The resulting cell cultures are consistently 100% pure a~ assessed by morphological and immunochemical criteria. The normal cells can be continuously pa~saged up to 10 times.
Vec~. The vector used in the transfection is dQsignated as SV-40T. It has the sequence that codes for the transformation protein of SV-40 large T antigen and RSV-LTR cloned into the Eco Rl site of the PBR322 plasmid.
~:lleSTlTUTE SHEET
.. . : . .: . , ' ,. . '~ ', ' .. : - -. .
W092~17569 PCT/US92/0~99 2 ~
Transfection. Primary human foreskin endothelial cells were ln their 6th passage from isolation when transfected. The procedure followed has been described ~y Graham and van der EB (Virology, 52:456-467, 1973). Some minor modifications were introduc~d to this ~rocedure. All surfaces to which endothelial cells attach were pre-coated with a solution of 0.1~ gelating in 0.01~ phosphate buffer saline ?h 7 a (73S) . ~he colls were plated onto 6-well plates a, 3.5 x lCs celia/-~ell and incuba-ced @
37C and 5~ C0. overnight ~e_ore transfection. The amount or vector DNA ~sed ~as ~ ~g per well.
Following ihe transfection ~rocedure, the plates were incubated overnight ~ 37C with 5% C0" then the lS contents of each well transferred to individual 25 cm2 plastic flasks with 0.2 ~ filter cap (Costar).
- Some of the flasks contained dibutyryl cAMP 0.5mM
(final concentration). Flasks were split 1:4 as they became confluent.
.
Detection of SV-40T viral antiqen. To detect expression of the SV-40T in the immortalized endothelial cells (C~C/EU.HMEC-1) an enzyme-linked immunoassay was performed. Cell line SV-T2 a Balb/3T3 mouse embryo line infected with SV-40 (ATCC
# CCL 163.1) was used as a positive control, a peripheral lymphocyte lysate was used as a negative control. Cell-free lysates were obtained by adding 0.5 ml of 0.5% deoxycholate.HCl in O.OlM P8S pH 8.2 and 100 ~l of a 1 mM solution of phenylmethyl sulfuflouride in 95% ethanol to 10 million cells.
This mixture was incubated Q 4C for 30 minutes and then centrifuged @ 4000 RPM for 15 minutes @ 4C.
.
. :
'. SU2STlT~iTE SHEET
.
~, ' ':' ". `
.. . ~ . . - - -. . .... ... ....
WO92/17569 PCT/US92/0~99 21Q7~9 The supernatant which possibly contained the SV-40 large T antigen was removed and saved.
For the ELISA test, the antigens were attached by suctioning onto nitrocellulose paper using a ~inifold II Slot Blotter (Schleifher &
Schuell, ~eD~e, NH). After the attachment procedure, the sheets were incubated for 24 hr with 2% s'clm mll'i; ~ 0.01 ~ PRS Q 4C to prevent nonspDcific i~inding. Afterwards, the skim mil~
0 solu- o.. is ~~-o-ta zr,d the ni.-ocellulose washed three tlmDs--iith 0.1~ TwePn-20 in PBS Q RT . ~ouse moncc'^r.21 a..__~o~ to .he S'v-40 large T viral antisen was added a~ a dilution of 1:1000 in PBS/Tween and incubated for 30 min @ 37C on an orbital shaker. The monoclonal antibody to SV-40T
antigen was obtained from the supernatant of the mouse hybridoma cell line Pab 101 (ATCC # TIB 117).
The nitrocellulose sheets were then washed three --times with Tween/PBS and an additional wash @ 37C
on the orbital shaker for 15 min. Goat anti-mouse - horseradish peroxidase labelled monoclonal Ab (Bio~ad, Ric~mond, CA) was added at a 1:2000 dilution and allowed to incubate for 30 minutes Q
37C shaking. Later, the nitrocellulose sheet was washed three times with Tween/PBS. A
diaminobenzidine (Sigma Chemical Co., St. Louis, MO) (DAB) solution consisting of 25 mg of DA~, 50 ml of PBS, and 20 ~l of 30i% HtO, was added and color allowed to develop. The nitrocellulose sheet was rinsed with deionized water to stop the enzymatic reaction.
, ~' ~E~ t ~UTE Si JE~
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,: ' , , '' ' ' ': . .'. .'~ ' :' ' '~"' ' ' ' ; '', ,`:'/ ,' . .' . :. . : : '. ; : ' .:
W092/17~69 PCTtUS92/0~99 2t~7~ '9 Expression of SV-40T antiaen. Cell-free lysates of CDC/EU.HMECI and SV-T2 (positive control) cells expressed the SV-40T viral antigen by ELISA and Western Blot. Negative cont~ol (hu~an peripheral blood lymphocyte lysate) cells were negative.
Characterization of endothelial cell culturPs.
Representative cultures of ~D;IEC and SV 4OT .iDMEC
(CDC/EU.HMEC-1) are charact2rized in thre~ ways.
Cultures are evaluate~ by nve-.ad has--con'_2~' microsccpy to d2tormine whethor 'h~ c_lls have he characteristic cob~laston2 .~or?hology e~^ ~ndotheliai cells. Cells are fixed in 90~ methanol at -20C for 10 minutes, washed and stained with a 1:40 dilution of rabbit anti-human factor VIII (Atlantic Antibodies; Scarborough, ME) for 30 minutes followed by FITC conjugated goat anti-rabbit IgG. They are washed three times and then viewed under a fluorescent microscope. Cells, unfixed, are incubated with acetylated low-density lipoprotein (10 ~g/ml), labeled with 1,l'dioctadecyl-1,3,3,3'3'-tetramethyl indocarbocyanine perchlorate (Dil-AC-LDL) (Biomedical Technology, Inc.; Stoughton, MA) at 37C in medium 199 without growth supplement or fetal calf serum for 4 hours. Dil-Ac-LDL is a biologic probe incorporated by living endothelial cells and, to a lesser extent, by monocytes or macrophages. The media is then removed, and the cells are washed twice and visualized in a fluorescence microscope with standard rhodamine excitation emission filters. Evaluation by all three techniques reveals that pure cultures of both types of endothelial cells are routinely attained.
SU~STITUTE SHEET
,. . - .:
W092tl7569 PCT/US92/0~ ~
2 ~ 5 9 Diffe Qr;tla.ion OL endotheli~l cells. Matrigel, an extrac. o h~ ~-IS sarcoma that contains basement membran~ co-~or.ents (Collaborative Research;
Bedford, MA), is applied to 24- or 96-well cell-cul.ur~ pla.2s as eit.~e a ch-cX or thin film and then incubat_d at 37C. This temperature induces selli~g ~ e ~t act. ~:DMEC or CDC/~U.HMEC-1 are th~r ~la~ . 3 '~ m_=_ igQ~ e cells at.ach rapid ~, ~ilG -~ n i-^~ ~OU_ S elongated process2s lo are 9bs~ d, and æ-^ter 8 hours the endothelial cell cul.u;^~s snow a~undant nP works of branching and anastomosing co~ds or cells. Bv light microscopy, most of the cords show a central translucent structure along their long axis, which suggests the presence of a lumen. By 8 hours, the endothelial cells form an interconnected network of anastomosing cells that by low-power light microscopy have a "honeycomb" appearance. These endothelial cells express factor VIII-related antigen before, during, and after tube formation. They are also metabolically active, since they ta~e up acetylated low-density lipoprotein. Transmission electron microscopy of cells cultured on matrigel for 18 hours demonstrates that cross-sections of the tube-like structures contain a lumen surrounded by cells.The membranes of the cells forming the lumen of the tubes connect with one another by interdigitating cytoplasmic processes.
Flow cytometry. Analysis of cell-surface molecules on HDMEC and CDC/EU.HMEC-1 was performed using direct immunofluorescence and flow cytometry.
SUBSTITUTE SHEET
: - - - . ., .. : , - - . . ~` -; .. . .. - -.. .
,. - . . . - . . ~ . , ~ .. . .
- , - . :,. . :- : : ,; , : :
... . . ..
WO92/17569 PCT/US92~0~ ~
2~7~ 9 Cytometric analysis was performed on a FACscan Flow Cytometer (3ecton-Dickinson, I~c.; ~ountain View, CA). This instrument provides data regarding cell number, forward angle light scatter, side scatter, and red and green fluorescence. Approximately 10,000 cells per test sample were analyzed in these studies. HDMEC and CDC/EU.HMEC-1 to be analyzed in these studies will be removed from tissue-culturo flas~s with 6-10 ml of 5~M Er~.~ and '~ 3S.~ to a~ d any loss of trypsin or dispase-sensi~ e enao~heliai cell epitopes. After incu~ating ror 30 minutes at 37C, an equal volume or HBSS with C~T~ and Mg+- is added to inactivate the EDTA, and the cells are washed three times. The cells are separated into aliquots of 10~ cells/tube, pelleted, supernatant discarded, and 20 ~1 of undiluted monoclonal antibody is added. The cells are lightly vortexed and incubated for 30 minutes on ice. The cells are washed three times and then either stained with an appropriate second-step antibody or ana-lyzed directly in one-step staining procedures.
Phenotv~ic Characterization of SV 40T HDMEC
The cell line, CDC/EU.HMEC-l is in its 40th passage. Control cells which did not contain SV-40T DNA or cAMP growth supplement after tran~fection died off after two additional passages after transfection. CDC/EU.HMEC-1 assumed a "cobblestone" morphology when cultured on gelatin-coated tissue culture dishes when cultured incomplete HDMEC media. Their morphology was essentially indistinguishable from HDMEC (Figure 1).
SU.~, T i ~ UTE SHcFT
. - . ' . . . ~ - . - . . . . .
i . . . . ~ .
.. . . . . . . ~ .
.. . - . . . . -. . - . -......... ..
- .. .. ,. ... . . - . . .. ..
, - . . . ~ . . . ~ . .
.. - - . . . . . - - . . . . .. , , . -WO92/17569 PCT/US92/0~ ~
21075a9 It was noted, however, that when allowed to become hyperconLluent, the CDC/EU.HMEC-l were capable of growing to a higher density and that the cells appeared nat~.al;y smalier t~an H3~EC under these conditions.
C3C~-TJ.~EC-' ar.à HDYEC both s.ained positively fo- Factor VIII wh_n examined by direct immunofluorDscer.~ cth yres 3_ c~lls also demons.-a~ ?-''~ c. aa_;ila.2d iow aensi -y lipopro~ein a^~ no ~s e~osur~ (~isure 2).
In or-er .o det-r~ine whe-her CDC/Eu.X~EC-1 were c2pabl ~ o~^ mor~h^logic di.ferentiation into tubes, th~se cDlls w~re cultured on m2trigol. It was previously shown that HDMEC will form capillary-like structures when cultured on the basement - ~ -membrane-line matrix (Xubota, Y., et al. (1988) J.
Cell Biol. 167:1589). CDC/EU.HMEC-l demonstrated tube formation after 8 hours of culture on matrigel which was indistinguishable from that of HDMEC
(Figure 3).
P~oliferation Studies It was previously demonstrated that optimal growth of HDMEC requires specialized growth medium supplemented with 30% human serum (Kubota, Y., et al. (1988) J. Cell Biol. 107:1589 and personal observation). Decreasing human serum to 20% or below essentially halts proliferation. In order to determine human serum requirements for CDC/EU.HMEC-l, comparison growth studies were performed with routine HDMEC medium supplemented with 30%, 20%, 10%, 5%, or 1% human serum. The results showed that although the cells grew best at ~UBSTIT'i .~
.. .. . . . .
- . : . ; . . . .
2 - .
. .
.: - :. - ` ' : .
- ' , . .
, . , ' . .. ' - ` . . ..
WO92~l7s69 PCT/US92/0 2 1 ~ 7 rj ~ 9 30% human serum, there was growth even at a concentration of 1% human serum (Figure 4).
Population doubling time ranged from 53.6 hours for cells cultured in media containing 30~ human serum to 85.6 hours for cells cultured in media containing 1% human serum. Further studi~os ex2mining srs~th curves of CDC/EU.HMEC-1 showed that CDC/EU.~IEC-1 were capable of replicating in routine HDM~C cul~uro media devoid of serum (Figure 5 ).
Cell Surface Molecule Ex~rsssion Previous studies have shown that HDMEC s~r~ss a number of cell adhesion molecules on their surface including ICAM-1, LFA-3, CD44, and Class I, but lack 15 constitutive expression of Class II molecules (Fleck, R. et al. (1986) J. Invest. Dermatol.
86:475). HDMEC and CDC/EU.HMEC-1 were compared for expression of these cell surface antigens. Both normal and transfected cells expressed ICAM-l, LFA-20 3, CD44, and Class I, but did not express Class II
(Table 1). However, stimulation of HDM~C and CDC/EU.HMEC-1 with IFN-gamma (500 ~/ml, 72 hrs) resulted in induction of Class II cell surface antigen expression (Figure 6). The CDC/EU.HMEC-1 25 expressed more than 3 times as much CD44 and more than twice as much ICAM-1 and LFA-3 than did nontransfected HDMEC. Class I expression was roughly equivalent on both types of cells.
T~3LE I. Comparison of expression of selected immunologically relevant cell-surface antigens on untransformed and CDC/EU.HMEC-... . . . . . . . . . . .
, , " - '' ' ; ' ''", ' '' ~ . , ",', ':' ~ ".,', .' ', . ' : ' ,. : ., ' , . , : : : ' , , :' . .: .
:" ' , , ', . ' ' . .' ' :- , ' ' : . :,, ' -: . ' . :
W092tl7569 PCT/US92/0~99 2.~ ~7~5~
1 human dermal microvascular endothelial c211s.
XLi'~EC COC~,;U. ~IEC-l IC~-l + +
L~A- 3 _ , CD4 4 +
Class II - - -Class TT~
~a.'e. stimu'a ion Fo~ n ~.~Jlth 500 u~ml o,^ l~-ga~ma.
* * * * *
All publications mentioned hereinabove are hereby incorporated in their entirety by reference.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.
S!J~,TUTE SKEEl r . ~
,, , , .,' .' . ' ' ' '- ,' ,.' ' , ' ' ,',, ~ " . .
, ' ' ~', '' ' ' -, ;i" ';,, , ,. : ~ , .. .
~''' . :- .' ` ' , ~' ' . .-
Claims (9)
1. A microvascular endothelial cell wherein said cell is obtained from primate skin and is immortalized.
2. The microvascular endothelial cell according to claim 1 wherein said primate skin is human skin.
3. The microvascular endothelial cell according to claim 1 wherein said human skin is human foreskin.
4. The microvascular endothelial cell according to claim 1 wherein said cell contains DNA
that encodes SV40 large T antigen.
that encodes SV40 large T antigen.
5. The microvascular endothelial cell line CDC/EU.HMEC-1, ATCC # CRL 10636.
6. A method of establishing a cell line of immortalized microvascular endothelial cells derived from primate skin comprising:
(1) introducing DNA which encodes SV40 large T
antigen into said cells under conditions such that said cells become immortalized and (2) culturing said cell line.
(1) introducing DNA which encodes SV40 large T
antigen into said cells under conditions such that said cells become immortalized and (2) culturing said cell line.
7. The method of establishing a cell line according to claim 5 wherein said primate skin is human skin.
8. The method of establishing a cell line according to claim 6 wherein said human skin is human foreskin.
9. The method of establishing a cell line according to claim 5 wherein said introducing DNA is executed by transfection.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US67967491A | 1991-04-04 | 1991-04-04 | |
| US679,674 | 1991-04-04 | ||
| PCT/US1992/002499 WO1992017569A1 (en) | 1991-04-04 | 1992-04-03 | Immortalization of endothelial cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2107559A1 true CA2107559A1 (en) | 1992-10-05 |
Family
ID=24727880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002107559A Abandoned CA2107559A1 (en) | 1991-04-04 | 1992-04-03 | Immortalization of endothelial cells |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0578769A4 (en) |
| JP (1) | JPH06509467A (en) |
| AU (1) | AU1765992A (en) |
| CA (1) | CA2107559A1 (en) |
| WO (1) | WO1992017569A1 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5133736A (en) * | 1990-05-10 | 1992-07-28 | Symbiosis Corporation | Investment cast end effectors for disposable laparoscopic surgical instrument |
| FR2681609B1 (en) * | 1991-09-24 | 1994-12-30 | Centre Nat Rech Scient | LINES OF IMMORTALIZED CEREBRAL ENDOTHELIAL CELLS, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS A STUDY MODEL OF CEREBRAL PHYSIOPATHOLOGY. |
| FR2726005B1 (en) * | 1994-10-10 | 1997-01-03 | Adim | IMMORTALIZED LINES OF CEREBRAL ENDOTHELIAL CELLS AND THEIR APPLICATIONS FOR THE TREATMENT OF DIFFERENT NEUROLOGICAL OR PSYCHIATRIC PRIMARY AND SECONDARY DISORDERS OR DISEASES |
| EP0893493A3 (en) * | 1997-07-21 | 2002-12-04 | Aventis Pharma Deutschland GmbH | Genetically modified cells and their use for prophylaxis or treatment of diseases |
| FR2787464B1 (en) * | 1998-12-21 | 2003-01-10 | Neurotech | PHARMACEUTICAL COMPOSITIONS COMPRISING IMMORTALIZED ENDOTHELIAL CELLS FOR THE DETECTION AND / OR TREATMENT OF ANGIOGENIC SOURCES AND PARTICULARLY CANCERS |
| DE19903920B4 (en) * | 1999-02-01 | 2005-08-25 | Zouboulis, Ch.C., Priv.-Doz.Dr. | Sebocytes, sebocyte cell line and their uses |
| AU4025100A (en) * | 1999-03-24 | 2000-10-09 | Board Of Trustees Of The Leland Stanford Junior University | Immortal microvascular endothelial cells and uses thereof |
| DE69926053T2 (en) * | 1999-04-27 | 2006-04-27 | Transgene S.A. | Process for the preparation of mammalian cell lines |
| EP1048724A1 (en) * | 1999-04-27 | 2000-11-02 | Transgene S.A. | Human myoblast cell lines and their uses |
| JP4321936B2 (en) * | 2000-02-16 | 2009-08-26 | 独立行政法人科学技術振興機構 | Immortalized pericyte cell line |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0272866A (en) * | 1988-09-07 | 1990-03-13 | Banyu Pharmaceut Co Ltd | Human vascular endothelical cell-derived transforming cell |
-
1992
- 1992-04-03 CA CA002107559A patent/CA2107559A1/en not_active Abandoned
- 1992-04-03 EP EP92910683A patent/EP0578769A4/en not_active Withdrawn
- 1992-04-03 AU AU17659/92A patent/AU1765992A/en not_active Abandoned
- 1992-04-03 JP JP4509699A patent/JPH06509467A/en active Pending
- 1992-04-03 WO PCT/US1992/002499 patent/WO1992017569A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1992017569A1 (en) | 1992-10-15 |
| EP0578769A4 (en) | 1995-08-16 |
| AU1765992A (en) | 1992-11-02 |
| EP0578769A1 (en) | 1994-01-19 |
| JPH06509467A (en) | 1994-10-27 |
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