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  • 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
  • C12N5/0679—Cells of the gastro-intestinal tract
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  • 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
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/05—Inorganic components
  • C12N2500/10—Metals; Metal chelators
  • C12N2500/20—Transition metals
  • C12N2500/24—Iron; Fe chelators; Transferrin
  • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/11—Epidermal growth factor [EGF]
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
  • C12N2501/39—Steroid hormones
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
  • C12N2501/39—Steroid hormones
  • C12N2501/392—Sexual steroids
• • 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
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening

Definitions

• the present invention is directed to a media formulation that allows the development of a highly differentiated intestinal epithelial cell monolayer in a much shorter period of time than currently possible without the aid of cell culture substrates.
• the invention is also directed to a method of culturing the cells, as well as a process for preparing the specialized media. This is a particular advantage for researchers who rely on such cell lines in studying the intestinal tract, and more particularly for the pharmaceutical industry, which uses these cell lines as models for drug absorption in the intestine.
• Intestinal epithelium is composed of a monolayer of morphologically polarized cells, which function in absorbing substances from the intestine and transporting them ultimately to the bloodstream.
• Several epithelial in vitro cell culture systems have been established in the past few decades, which mimic the cells lining the intestine.
• Some of the cell lines are Caco-2, HT-29, SW 1116, T84, IEC-18, and IEC-6.
• Caco-2 and HT-29 intestinal cell lines are the most widely used and best characterized systems.
• Other experimental models that have been under investigation include various clones of these lines (e.g., Caco- 2/TC-7 (Caro et al., Int. J.
• Caco-2 cells are the cells of choice for drug transport research and analysis. These cells differentiate spontaneously in culture some time after reaching confluence. Caco-2 cells usually require about 14 - 30 days after reaching confluence, with an average of 21 days post-confluence, to fully differentiate, i.e. differentiate to the point at which they exhibit many of the morphological, enzymatic, and most importantly, the nutrient transport characteristics of normal human intestinal cells. This is a comparatively long time to wait for cells to be ready to perform experiments, and requires thoughtful long-term planning for conducting studies with this system.
• the cells are seeded on the Biocoat® Fibrillar Collagen Cell Culture Inserts (Becton Dickinson) and cultured in the specialized medium supplemented with butyric acid, hormones, growth factors and other defined metabolites.
• An accelerated Caco-2 model has been developed using a novel media formulation that allows development of highly differentiated Caco-2 cell monolayer in about 4 days, as compared to the traditional 21 -day culture.
• the media formulation of the present invention contains certain supplements that, when used together, surprisingly provide an environment conducive to differentiation.
• the supplements added to the cell culture medium are fetal bovine serum, nonessential amino acids, transferrin (preferably human), insulin (preferably bovine), epidermal growth factor (preferably human or mouse), sodium butyrate or butyric acid, and the hormones hydrocortisone, progesterone and testosterone.
• the present invention is also directed to a process for preparing the medium, as well as a method for using the medium to obtain accelerated growth of an intestinal cell line.
• An object of the present invention is to provide a composition for culturing intestinal epithelial cell lines, which contains a cell culture growth medium supplemented with fetal bovine serum, nonessential amino acids, human transferrin, bovine insulin, human epithelial growth factor, butyric acid or salts thereof, hydrocortisone, progesterone, and testosterone.
• Previous investigations of rapid Caco-2 models revealed that the accelerated differentiation of intestinal epithelial cells is triggered by the synergistic action of nutrients and growth factors contained in a culturing medium.
• Insulin, epidermal growth factor (EGF), transferrin and various hormones are used routinely as media supplements that facilitate protein and amino acid synthesis, phosphate transport, lipogenesis and cell proliferation. Addition of these and other supplements to cell culture media is especially important when cells are grown in reduced-serum and serum-free conditions. While the exact composition of serum is not defined, it is known to contain a variety of components needed for cell growth and differentiation. For example, insulin provides signals for cell multiplication, while transferrin, EGF and hormones promote cell differentiation (Chopra et al., 1987, Gastroenterology, 92; Souleimani and Asselin, 1993, FEBS Lett., 326).
• the concentrations of supplements in the media for intestinal epithelial cells can vary somewhat and some amount of optimization for a particular intestinal cell line is to be expected. In general, however, the medium concentrations of insulin, EGF, and transferrin range from 0.01 to 200 ⁇ g/mL, while the content of hormones such as progesterone, testosterone, and hydrocortisone should fall between 0.01 and 10 ⁇ M.
• Butyric acid or its salts (such as sodium butyrate), a known differentiation agent, will be effective at concentrations ranging from 0.5 to 5 mM (Siavoshian et al., 1997).
• the medium composition is considered optimal when it yields TEER (transepithelial electrical resistance) values of about 200 Ohm x cm 2 and above in 4-day-old Caco-2 monolayers that are seeded on regular polycarbonate filters without collagen support (Transwell®, Corning- Costar).
• the formulation of the specialized media is based on any of several known cell culture media, and the choice of which one to use should reflect the recommended growth medium for the particular cell line being grown.
• the media used for development of the accelerated Caco-2 cell model of the present invention was based on DMEM/F-12 medium supplemented with fetal bovine serum and about 1% nonessential amino acids.
• Other media could be, for example McCoy's 5a (for HT-29), Eagle's minimal essential medium (for some Caco-2 lines), or RPMI.
• the media should also contain a source of I- glutamine, typically about 2 to about 4 mM.
• the concentration of fetal bovine serum is from about 5 to about 20% of the medium formulation.
• transferrin available, for example from VWR
• this can be a human, bovine or mouse form, but preferably is human.
• the epidermal growth factor either human and mouse can be used interchangeably, although preferred is human.
• a preferred specialized accelerated growth medium (named "HTB- 10") was prepared for Caco-2 cells by supplementing DMEM/F-12 medium with 10% fetal bovine serum, 1% nonessential amino acids, 100 ⁇ g/mL human transferrin, 30 ⁇ g/ml bovine insulin, 50 ng/mL human epidermal growth factor (EGF), 2 mM sodium butyrate, and 5 ⁇ M of each hydrocortisone, progesterone, and testosterone.
• the medium optionally, but preferably, contains one or more antibiotics, such as penicillin (at about 100 U/ml), streptomycin (at about 100 ⁇ g/ml) and amphotericin B (at about 0.25 ⁇ g/ml).
• a method of using the medium also is part of the present invention. The following description is representative of how this would be done.
• Caco-2 cells (available from ATCC, Manassas, VA) are resuspended in the HTB-10 medium and seeded on 3 ⁇ m polycarbonate 6-well Transwell ® cell culture inserts (diameter 24.5 mm, Corning Costar) at a density of about 0.2 x 10 6 cells/cm 2 .
• the seeding of the cells was performed on dry filters by placing 1.5 mL of the cell suspension on the apical side first, followed by addition of 2.5 mL of HTB-10 medium to the baso-Iateral side.
• the cells are grown in a 37°C incubator at about 95% relative humidity with about 5% CO 2 .
• the advantages of the invention include: (1 ) decreased time for obtaining differentiated Caco-2 monolayers for permeability assays; and (2) reduced cost and time of cell culturing and maintenance through the use of ordinary Transwell® plates without collagen support and by reduction of incubation time in culture.
• the new media of the present invention has allowed for the development of an alternative experimental system with characteristics compatible with traditional 21 -day Caco-2 cell model.
• the Use of the specialized accelerated growth medium HTB-10 allowed us to obtain a differentiated Caco-2 monolayer in a 4-day period.
• the differentiation status and the monolayer integrity were monitored by measurements of transepithelial electrical resistance (TEER) and by determination of permeability of mannitol.
• Average TEER developed after 4 day culturing in HTB-10 ranged between 420 to 1090 Ohm x cm 2 .
• the permeability of mannitol ranged from 0.7 x 10 "6 to 6.3 x 10 "6 cm/sec.
• the accelerated Caco-2 system was also evaluated through a validation process that was based on FDA guidelines for the Biopharmaceuticals Classification System (BCS).
• BCS Biopharmaceuticals Classification System
• Caco-2 cell permeability coefficients were determined for 26 structurally diverse compounds that were then rank-ordered according to the fraction absorbed in humans.
• the transport studies were conducted as follows. Drug solutions were prepared in HBSS (pH 7.4) at final concentrations indicated in Table 1. The donor solution of the drug was placed on the apical side of a sample filter (1.5 mL) and the buffer solution (HBSS, pH 7.4) was placed on the baso-Iateral side (2.5 mL). The plates were incubated at 37°C on a shaker for 1- 3 hr. Samples were collected at the designated time points and analyzed by either HPLC (uv)or radiometry (for radioactive compounds). The suitability of the accelerated Caco-2 system was validated by determination of permeabilities of several marker compounds including methotrexate, propranolol, and testosterone.
• Table 1 Permeability of the model compounds used for validation of the accelerated Caco-2 system.
• FIG. 1 The Table 2, below, summarizes the media composition of three comparative systems: Biocoat, Lentz et al., and that of the present invention.
• Figure 1 is a graph obtained from the comparison study of the different types of media. Depending on the medium, the seeding was done as described in our procedure (for HTB-10, HTB-0), or according to the Biocoat instructions, or according to the procedure described in Lentz's paper (for HTB-37). The TEERs were measured on the 4th day post-seeding. As can be seen from Figure 1 , the medium of the present invention (in this case, HTB-10) outperformed all of the others in non-collagen supported cell differentiation.

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• 2004
  • 2004-03-11 JP JP2006507065A patent/JP2006520598A/ja active Pending
  • 2004-03-11 US US10/797,952 patent/US7300794B2/en not_active Expired - Fee Related
  • 2004-03-11 CA CA002518496A patent/CA2518496A1/en not_active Abandoned
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