Hormone responsive tissue culture system and uses thereof

Abstract

The invention provides tissue culture system for primary cells (e.g. normal mammalian primary epithelial progenitors). This system includes: a) a serum-free, chemically defined cell culture media; and, b) methods for isolation and in vitro long-term propagation of primary cells (e.g. primary epithelial cells). Primary cells so isolated and cultured can be kept undifferentiated and proliferate for many weeks (>15 weeks) or population doubling (>35 PD) without senescence, or any detectable genetic alterations. Upon changing media / culture conditions, these cells can be induced to differentiate.The invention also provides methods to transform normal primary cells so cultured into “cancer stem cells.” The genetically defined cancer stem cell tumor model mimics the behavior of the disease closely, e.g., the cells are invasive, hormone responsive and metastatic when injected into mice. The tumor cells express genes that are specific to cancer stem cells identified in patient samples.

Description

RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. provisional application 60 / 964,271, entitled “Hormone Responsive Tissue Culture System and Uses Thereof,” filed Aug. 10, 2007. This application is related to U.S. provisional application 60 / 569,005, entitled “Contribution of Target Cell Type to Epithelial Tumor Phenotypes,” filed May 7, 2004 and U.S. provisional application 60 / 630,934, entitled “Hormone Responsive Tissue Culture System and Uses Thereof,” filed Nov. 24, 2004. The entire teachings of the referenced applications are expressly incorporated herein by reference.GOVERNMENT SUPPORT

[0002] The invention described herein was supported, in whole or in part, by Grant No. K08-CA-92013 from the National Cancer Institute (NCI). The U.S. Government has certain rights in this invention.BACKGROUND OF THE INVENTION

[0003] Recent work comparing transformation and tumorigenicity of rodent and human cells has established significant differences between s...

Examples

examples

[0353]The following examples are for illustrative purpose only, and should not be construed to be limiting in any respect of the claimed invention.

example i

Isolation of Primary Cells

[0354]1. Tissues are minced down to fragments about 1-2 mm in size, and digested with collagenase in Hank's buffered saline solution (HBSS) at 37° C. overnight.[0355]2. The resulting mixture of organoids that contain glandular cells are separated from single cells that are mostly stromal and myoepithelial by three consecutive rounds of centrifugation (5 min. at 300×g, 100×g and 50×g).[0356]3. The pellets and supernatants are collected in six fractions (300 g, 100 g and 50 g) and filtered through a 75 micrometer (μm) and 45 μm mesh.[0357]4. All filtered fractions are plated in Primaria™ tissue culture dishes in primary culture medium PWI (see below).[0358]5. The cells are grown for 7 days during which medium is changed every day.[0359]6. On day 8 and 9 plates are trypsinized with 0.025% trypsin removing all stromal cells.[0360]7. Epithelial cells are harvested with 0.15% trypsin and transferred to new Primaria™ plates in PWI medium.[0361]8. After one week ce...

example ii

Methods of Preparing the Media

[0363]The media disclosed herein can be made fresh every time from their individual components, which are commercially available from a variety of vendors, such as Sigma, Abott Lab., etc.

[0364]Alternatively, certain components of the media may be pre-made as high concentration stock solutions, which can be diluted to their final concentrations as listed in the Tables. The stock solutions should be appropriately stored according to the characteristics of the components, including stability at the storage temperature (e.g., liquid nitrogen, −80° C., −20° C., 4° C., room temperature or about 20-25° C., etc.), sensitiveness to light, natural half life in aqueous or organic solution, etc. Some stock solutions should be remade periodically to keep a fresh stock. The following lists at least one way of preparing several exemplary stock solutions. Other equivalent methods and similar (but not identical) concentration of stock solutions may also be used.

EGF (Epi...

RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. provisional application 60 / 964,271, entitled “Hormone Responsive Tissue Culture System and Uses Thereof,” filed Aug. 10, 2007. This application is related to U.S. provisional application 60 / 569,005, entitled “Contribution of Target Cell Type to Epithelial Tumor Phenotypes,” filed May 7, 2004 and U.S. provisional application 60 / 630,934, entitled “Hormone Responsive Tissue Culture System and Uses Thereof,” filed Nov. 24, 2004. The entire teachings of the referenced applications are expressly incorporated herein by reference.GOVERNMENT SUPPORT

[0002] The invention described herein was supported, in whole or in part, by Grant No. K08-CA-92013 from the National Cancer Institute (NCI). The U.S. Government has certain rights in this invention.BACKGROUND OF THE INVENTION

[0003] Recent work comparing transformation and tumorigenicity of rodent and human cells has established significant differences between s...

Examples

examples

[0353]The following examples are for illustrative purpose only, and should not be construed to be limiting in any respect of the claimed invention.

example i

Isolation of Primary Cells

[0354]1. Tissues are minced down to fragments about 1-2 mm in size, and digested with collagenase in Hank's buffered saline solution (HBSS) at 37° C. overnight.[0355]2. The resulting mixture of organoids that contain glandular cells are separated from single cells that are mostly stromal and myoepithelial by three consecutive rounds of centrifugation (5 min. at 300×g, 100×g and 50×g).[0356]3. The pellets and supernatants are collected in six fractions (300 g, 100 g and 50 g) and filtered through a 75 micrometer (μm) and 45 μm mesh.[0357]4. All filtered fractions are plated in Primaria™ tissue culture dishes in primary culture medium PWI (see below).[0358]5. The cells are grown for 7 days during which medium is changed every day.[0359]6. On day 8 and 9 plates are trypsinized with 0.025% trypsin removing all stromal cells.[0360]7. Epithelial cells are harvested with 0.15% trypsin and transferred to new Primaria™ plates in PWI medium.[0361]8. After one week ce...

example ii

Methods of Preparing the Media

[0363]The media disclosed herein can be made fresh every time from their individual components, which are commercially available from a variety of vendors, such as Sigma, Abott Lab., etc.

[0364]Alternatively, certain components of the media may be pre-made as high concentration stock solutions, which can be diluted to their final concentrations as listed in the Tables. The stock solutions should be appropriately stored according to the characteristics of the components, including stability at the storage temperature (e.g., liquid nitrogen, −80° C., −20° C., 4° C., room temperature or about 20-25° C., etc.), sensitiveness to light, natural half life in aqueous or organic solution, etc. Some stock solutions should be remade periodically to keep a fresh stock. The following lists at least one way of preparing several exemplary stock solutions. Other equivalent methods and similar (but not identical) concentration of stock solutions may also be used.

EGF (Epi...

Images