- Email: [email protected]
Short-Term Primary Culture of Epithelial Cells From Human Colon
February 1989
EDITORIALS
carriers with HTLV-III antibodies to recombinant a-interferon. Hepatology 1987;7:719-23. 23. Bortolotti F, Calzia R. Vegnente A, et al. Chronic hepatitis in childhood: the spectrum of the disease. Gut 1988;29:659-64. 24. Peters M, Walling DM , Kelly K, Davis GL, Waggoner JG, Hoofnagle ]H. Immunologic effects of interferon-a in man : treatment with human recombinant interferon-a suppresses in vitro immunoglobulin production in patients with chronic type B hepatitis. ] ImmunoI1986 ;137:3147-52.
535
25. Scullard GH, Alberti A, Wansbrough-]ones MH, et al. Effects of human leucocyte interferon on hepatitis B virus replication and immune respons es in patients with chronic hepatitis B infection. ] Clin Lab Immunol 1979;1:277-82. Address requests for reprints to: R. Perrillo, M.D., Veterans Administration Medical Center 111 ]C, 915 North Grand Boulevard , St. Louis , Missouri 63106. © 1989 by the American Gastroenterological Association
Short-Term Primary Culture of Epithelial Cells From Human Colon The complexity of the intestinal mucosa, with the possibility of extensive interactions among different cell types, represents a formidable obstacle to the in vivo study of the factors that influence epithelial cell proliferation, induction and modulation of cell differentiation, and synthesis of tissue- and cell-specific products. The ideal model system for such studies would be a pure culture of epithelial cells that have retained in vitro the differentiated characteristics and the regulatory mechanisms operative in the same cells in vivo. Thus , much effort has been devoted in recent years to the culture of intestinal tissue and cells: various techniques have been described, and each of them has both advantages and limitations. Organ culture of adult intestinal fragments is limited in use by the rapid necrosis and degeneration of the epithelium; although quite useful for short-term metabolic studies, such cultures can be effectively maintained in vitro for a maximum of 12-24 h. They also fail to solve the problem represented by the cellular complexity of the tissue of origin. Better tissue preservation and longer survival times have been reported for fetal or embryonic rat (1) and chick (2) small intestine. Monolayer cultures of intestinal cryptlike cells have been established by various groups (3-6) and have been characterized extensively, but success in inducing their differentiation in vitro has been, to this date, limited (7). A high degree of differentiation could only be obtained by association of these cells with intestinal mesenchyme followed by grafting under the kidney capsule (6). Tumor colon cell lines, in particular Caco-2 (B,9), HT-29 (10), and TB4 (11,12) cells, represent useful model systems for the study of some intestinal activities (13), but their similarity with normal adult epithelial cells must be questioned because of their origin. There is, therefore, an urgent
need for a reliable primary culture system that can provide relatively pure, fully differentiated, epithelial cell populations, and the paper by Gibson et a1. (14) appearing in this issue represents an important step toward that goal. The major objective of this work was to develop a technique for isolation of relatively pure populations of colonic epithelial cells, and their primary explant culture, starting with resected mucosa or colonoscopic biopsy specimens. This procedure is based on two main steps: (a) isolation of epithelial sheets and whole crypts by digestion of mucosal scrapings with dispase and collagenase and (b) separation of the epithelial cells from mucus and undigested fragments of lamina propria by passage through a small needle followed by a coarse stainless steel mesh. The final cell population consists mainly of entire crypts and crypt fragments , with few single cells present. The isolated cell population was characterized with respect to (a) light microscopic morphology, (b) ultrastructural features (examined by both transmission electron microscopy and scanning electron microscopy, (c) viability (cell membrane integrity) , and (d) ability to proliferate (DNA synthesis, estimated as incorporation of radioactive thymidine into cellular DNA). The epithelial nature of the cells was also evaluated by immunohistochemical staining using antibodies to cytokeratin 18 (specific for epithelial cells) , vimentin (present in fibroblasts , muscle, and endothelial cells) , a surface antigen of normal colonic epithelial cells (defined by antibody HuTuM2) , GAP B.3 (expressed by leukocytes). and la-like antigens. The average cell yield was estimated at about 13 x 10 6 cells/cm 2 of mucosa, with high enrichment for epithelial cells (excluding erythrocytes, only 6.5% of the cells did not express epithelial markers) . The
536 EDITORIALS
structural integrity of the isolated cells was very good, both in terms of maintenance of cell polarity with good preservation of microvilli on the luminal surface and excellent organelle and nuclear morphology. Cell viability, estimated by different methods (trypan blue exclusion, leakage of 5tCr from preloaded cells, and leakage of the cytosolic enzyme lactic dehydrogenase) was > 95%. Surprisingly, contamination by bacteria or other microorganisms was found to be negligible over 7 days of culture. Freshly isolated cells were placed in culture and monolayer sheets of cells were observed after overnight incubation. The monolayers, which often surrounded centrally located crypts, continued to increase in size for 3 days, but were progressively lost thereafter. Contamination by nonepithelial cell types, in particular fibroblasts, was very low. Linear incorporation of radioactive thymidine into DNA was demonstrated for at least 1-2 days of culture. The simplicity and reproducibility of this new method should allow its application to essentially all available intestinal specimens, provided that they can be processed soon after resection. The authors appear to have solved some of the major problems encountered by previous studies, namely contamination by nonepithelial cell types and microorganisms and limited cell viability. At the time they are placed in culture, the epithelial cells remain in the organized conformation typical of the colonic crypts in vivo, and this may help in preserving biologically important cell-cell interactions and differentiated cell functions. The demonstrated maintenance of cell morphology, epithelial polarity and markers, and proliferative activity over at least 3 days in culture should allow ample time for even complex manipulations and biosynthetic and other studies. Thus, this new procedure fills an important gap in our array of available experimental methods for the study of intestinal epithelial cell functions and interactions. Future studies will undoubtedly reveal the full potential of this technique, in particular with respect to the response of the epithelial cells to hormones and growth factors, the effects of extracellular matrix components on cell proliferation and differentiation, and the investigation of physiologically important colonic cell activities. These studies should also contribute to our knowledge of the intestinal mucosa in disease states such as cancer,
GASTROENTEROLOGY Vol. 96. No.2. Part 1
sprue, inflammatory bowel disease, and interaction of pathogens with the intestinal cell surface. ANDREA QUARONI. Ph.D.
Section of Physiology Cornell University Ithaca. New York
References 1. Quaroni A. Development of fetal rat intestine in organ and monolayer culture. J Cell Bioi 1985;100:1611-22. 2. Corradino RA. Embryonic chick intestine in organ culture: hydrocortisone and vitamin D-mediated processes. Arch Biochern Biophys 1979;192:302-10. 3. Quaroni A. Wands J, Trelstad RL. Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell BioI 1979;80: 248-65. 4. Chopra DP. Yeh KY. Long term culture of epithelial cells from the normal rat colon. In Vitro 1981 ;17:441-9. 5. Blay J, Brown KD. Functional receptors for epidermal growth factor in an epithelial cell line derived from rat small intestine. Biochem J 1985 ;22 5:85-94 . 6. Kedinger M. Simon-Assmann PM. Lacroix B. Marxer A. Hauri HP. Haffen K. Fetal gut mese nchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev BioI 1986;113:474-83. 7. Carroll KM. Wong TT. Drabik DL. Chang EB. Differentiation of rat small intestinal epithelial cells by extracellular matrix. Am J Physiol 1988;254:G355-60. 8. Rousset M. Laburthe M. Pinto M. et al. Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2 : modulation by forskolin. J Cell Physiol 1985;123: 377-85. 9. Huet C. Sahuquillo-Merino C. Coudrier E. Louvard D. Absorptive and mucus-secreting subcJones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell BioI 1987;105 : 345-57. 10. Zweibaum A. Pinto M. Chevalier G. et al. Enterocytic differentiation of a sub population of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose. J Cell Physiol 1985;122:21-9. 11. Dharmsathaphorn K. McRoberts JA. Mandel KG. Tisdale LD. Masui H. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am J PhysioI1984;246:G204-8. 12. Madara ]L. Dharmsathaphorn K. Occluding junction structure-function relationships in a cultured epithelial monolayer. J Cell Bioi 1985;101:2124-33. 13. Hauri HP, Sterchi EE. Bienz D, Fransen JA. Marxer A. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Bioi 1985;101 :838-51. 14. Gibson PRo Van de Pol E. Maxwell LE , Gabriel A. Doe WF. Isolation of colonic crypts that maintain structural and metabolic viability in vitro. Gastroenterology 1989;96:283-91. Address requests for reprints to: Andrea Quaroni. Ph.D .• Associate Professor. Section of Physiology. Cornell University. 820 Veterinary Research Tower. Ithaca. New York 14853. © 1989 by the American Gas troenterological Association
EDITORIALS
carriers with HTLV-III antibodies to recombinant a-interferon. Hepatology 1987;7:719-23. 23. Bortolotti F, Calzia R. Vegnente A, et al. Chronic hepatitis in childhood: the spectrum of the disease. Gut 1988;29:659-64. 24. Peters M, Walling DM , Kelly K, Davis GL, Waggoner JG, Hoofnagle ]H. Immunologic effects of interferon-a in man : treatment with human recombinant interferon-a suppresses in vitro immunoglobulin production in patients with chronic type B hepatitis. ] ImmunoI1986 ;137:3147-52.
535
25. Scullard GH, Alberti A, Wansbrough-]ones MH, et al. Effects of human leucocyte interferon on hepatitis B virus replication and immune respons es in patients with chronic hepatitis B infection. ] Clin Lab Immunol 1979;1:277-82. Address requests for reprints to: R. Perrillo, M.D., Veterans Administration Medical Center 111 ]C, 915 North Grand Boulevard , St. Louis , Missouri 63106. © 1989 by the American Gastroenterological Association
Short-Term Primary Culture of Epithelial Cells From Human Colon The complexity of the intestinal mucosa, with the possibility of extensive interactions among different cell types, represents a formidable obstacle to the in vivo study of the factors that influence epithelial cell proliferation, induction and modulation of cell differentiation, and synthesis of tissue- and cell-specific products. The ideal model system for such studies would be a pure culture of epithelial cells that have retained in vitro the differentiated characteristics and the regulatory mechanisms operative in the same cells in vivo. Thus , much effort has been devoted in recent years to the culture of intestinal tissue and cells: various techniques have been described, and each of them has both advantages and limitations. Organ culture of adult intestinal fragments is limited in use by the rapid necrosis and degeneration of the epithelium; although quite useful for short-term metabolic studies, such cultures can be effectively maintained in vitro for a maximum of 12-24 h. They also fail to solve the problem represented by the cellular complexity of the tissue of origin. Better tissue preservation and longer survival times have been reported for fetal or embryonic rat (1) and chick (2) small intestine. Monolayer cultures of intestinal cryptlike cells have been established by various groups (3-6) and have been characterized extensively, but success in inducing their differentiation in vitro has been, to this date, limited (7). A high degree of differentiation could only be obtained by association of these cells with intestinal mesenchyme followed by grafting under the kidney capsule (6). Tumor colon cell lines, in particular Caco-2 (B,9), HT-29 (10), and TB4 (11,12) cells, represent useful model systems for the study of some intestinal activities (13), but their similarity with normal adult epithelial cells must be questioned because of their origin. There is, therefore, an urgent
need for a reliable primary culture system that can provide relatively pure, fully differentiated, epithelial cell populations, and the paper by Gibson et a1. (14) appearing in this issue represents an important step toward that goal. The major objective of this work was to develop a technique for isolation of relatively pure populations of colonic epithelial cells, and their primary explant culture, starting with resected mucosa or colonoscopic biopsy specimens. This procedure is based on two main steps: (a) isolation of epithelial sheets and whole crypts by digestion of mucosal scrapings with dispase and collagenase and (b) separation of the epithelial cells from mucus and undigested fragments of lamina propria by passage through a small needle followed by a coarse stainless steel mesh. The final cell population consists mainly of entire crypts and crypt fragments , with few single cells present. The isolated cell population was characterized with respect to (a) light microscopic morphology, (b) ultrastructural features (examined by both transmission electron microscopy and scanning electron microscopy, (c) viability (cell membrane integrity) , and (d) ability to proliferate (DNA synthesis, estimated as incorporation of radioactive thymidine into cellular DNA). The epithelial nature of the cells was also evaluated by immunohistochemical staining using antibodies to cytokeratin 18 (specific for epithelial cells) , vimentin (present in fibroblasts , muscle, and endothelial cells) , a surface antigen of normal colonic epithelial cells (defined by antibody HuTuM2) , GAP B.3 (expressed by leukocytes). and la-like antigens. The average cell yield was estimated at about 13 x 10 6 cells/cm 2 of mucosa, with high enrichment for epithelial cells (excluding erythrocytes, only 6.5% of the cells did not express epithelial markers) . The
536 EDITORIALS
structural integrity of the isolated cells was very good, both in terms of maintenance of cell polarity with good preservation of microvilli on the luminal surface and excellent organelle and nuclear morphology. Cell viability, estimated by different methods (trypan blue exclusion, leakage of 5tCr from preloaded cells, and leakage of the cytosolic enzyme lactic dehydrogenase) was > 95%. Surprisingly, contamination by bacteria or other microorganisms was found to be negligible over 7 days of culture. Freshly isolated cells were placed in culture and monolayer sheets of cells were observed after overnight incubation. The monolayers, which often surrounded centrally located crypts, continued to increase in size for 3 days, but were progressively lost thereafter. Contamination by nonepithelial cell types, in particular fibroblasts, was very low. Linear incorporation of radioactive thymidine into DNA was demonstrated for at least 1-2 days of culture. The simplicity and reproducibility of this new method should allow its application to essentially all available intestinal specimens, provided that they can be processed soon after resection. The authors appear to have solved some of the major problems encountered by previous studies, namely contamination by nonepithelial cell types and microorganisms and limited cell viability. At the time they are placed in culture, the epithelial cells remain in the organized conformation typical of the colonic crypts in vivo, and this may help in preserving biologically important cell-cell interactions and differentiated cell functions. The demonstrated maintenance of cell morphology, epithelial polarity and markers, and proliferative activity over at least 3 days in culture should allow ample time for even complex manipulations and biosynthetic and other studies. Thus, this new procedure fills an important gap in our array of available experimental methods for the study of intestinal epithelial cell functions and interactions. Future studies will undoubtedly reveal the full potential of this technique, in particular with respect to the response of the epithelial cells to hormones and growth factors, the effects of extracellular matrix components on cell proliferation and differentiation, and the investigation of physiologically important colonic cell activities. These studies should also contribute to our knowledge of the intestinal mucosa in disease states such as cancer,
GASTROENTEROLOGY Vol. 96. No.2. Part 1
sprue, inflammatory bowel disease, and interaction of pathogens with the intestinal cell surface. ANDREA QUARONI. Ph.D.
Section of Physiology Cornell University Ithaca. New York
References 1. Quaroni A. Development of fetal rat intestine in organ and monolayer culture. J Cell Bioi 1985;100:1611-22. 2. Corradino RA. Embryonic chick intestine in organ culture: hydrocortisone and vitamin D-mediated processes. Arch Biochern Biophys 1979;192:302-10. 3. Quaroni A. Wands J, Trelstad RL. Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell BioI 1979;80: 248-65. 4. Chopra DP. Yeh KY. Long term culture of epithelial cells from the normal rat colon. In Vitro 1981 ;17:441-9. 5. Blay J, Brown KD. Functional receptors for epidermal growth factor in an epithelial cell line derived from rat small intestine. Biochem J 1985 ;22 5:85-94 . 6. Kedinger M. Simon-Assmann PM. Lacroix B. Marxer A. Hauri HP. Haffen K. Fetal gut mese nchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev BioI 1986;113:474-83. 7. Carroll KM. Wong TT. Drabik DL. Chang EB. Differentiation of rat small intestinal epithelial cells by extracellular matrix. Am J Physiol 1988;254:G355-60. 8. Rousset M. Laburthe M. Pinto M. et al. Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2 : modulation by forskolin. J Cell Physiol 1985;123: 377-85. 9. Huet C. Sahuquillo-Merino C. Coudrier E. Louvard D. Absorptive and mucus-secreting subcJones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation. J Cell BioI 1987;105 : 345-57. 10. Zweibaum A. Pinto M. Chevalier G. et al. Enterocytic differentiation of a sub population of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose. J Cell Physiol 1985;122:21-9. 11. Dharmsathaphorn K. McRoberts JA. Mandel KG. Tisdale LD. Masui H. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am J PhysioI1984;246:G204-8. 12. Madara ]L. Dharmsathaphorn K. Occluding junction structure-function relationships in a cultured epithelial monolayer. J Cell Bioi 1985;101:2124-33. 13. Hauri HP, Sterchi EE. Bienz D, Fransen JA. Marxer A. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Bioi 1985;101 :838-51. 14. Gibson PRo Van de Pol E. Maxwell LE , Gabriel A. Doe WF. Isolation of colonic crypts that maintain structural and metabolic viability in vitro. Gastroenterology 1989;96:283-91. Address requests for reprints to: Andrea Quaroni. Ph.D .• Associate Professor. Section of Physiology. Cornell University. 820 Veterinary Research Tower. Ithaca. New York 14853. © 1989 by the American Gas troenterological Association