Cell yield of synthetic tissue control and synthetic tissue microarray control

Abstract

Pathological and clinical laboratory testing are important aspects of modern diagnostic and prognostic practices. Control samples are often used to maintain quality control (QC) for the reproducibility of test results through immunohistochemistry (IHC) staining, in situ hybridization (ISH), and other molecular analysis methods. Some of the controls available for IHC and ISH staining of tumor and other diseased tissues are derived from cancer tissues. However, such types of controls are available in very limited quantities, and once they are exhausted, alternative controls with the same characteristics may not be available. Another type of available control is derived from cancer cell lines. However, such controls do not exhibit consistent patterns and levels of cellular expression of a given marker or the heterogeneity of said expression that is ubiquitous in tumor tissues. As a result, these controls bear little or no morphological resemblance to actual tumor tissues. Furthermore, conventional techniques for generating controls involve low-yield processes, requiring a significant amount of cultured cells to generate such controls, thereby increasing production costs and reducing production efficiency. [Selected Figure] Figure 1

Description

[Background technology]

[0001] Pathological and clinical laboratory testing are important aspects of modern diagnostic and prognostic practice. Control samples are often used to maintain quality control (QC) for the reproducibility of test results through immunohistochemistry (IHC) staining, in situ hybridization (ISH), and other molecular analytical methods.

[0002] Some of the controls available for IHC and ISH staining of tumor tissues and other diseased tissues are derived from cancer tissues. However, such types of controls are available in limited quantities, and once they are exhausted, replacement controls with the same characteristics may not be available. Another type of available control is derived from cancer cell lines. However, such controls do not exhibit consistent patterns and levels of cellular expression of a given marker or the heterogeneity of said expression that is ubiquitous in tumor tissues. As a result, these controls bear little or no morphological resemblance to actual tumor tissues. Furthermore, conventional techniques for forming controls involve low-yield processes, requiring a significant amount of cultured cells to form such controls, thereby increasing production costs and reducing production efficiency. The prior art documents relevant to the invention of this application are as follows (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent document) (Patent Document 1) U.S. Patent Application Publication No. 2001 / 0055804 (Patent Document 2) U.S. Patent Application Publication No. 2014 / 0142370 (Patent Document 3) U.S. Patent No. 5,153,132 (Patent Document 4) U.S. Patent Application Publication No. 2006 / 0148074 (Patent Document 5) U.S. Patent Application Publication No. 2011 / 0306110 (Patent Document 6) U.S. Patent No. 5,851,816 (Non-patent literature) (Non-patent document 1) Abcam "ab115347 - Live / Dead Cell Assay" 2012.pg 7 (Non-Patent Document 2) Roche Diagnostics GmBH "DNase I" December 2010. Application section Summary of the Invention

[0003] The disclosed embodiments provide methods for forming synthetic tissue controls and synthetic tissue microarray controls for IHC and ISH tests for cancer diagnosis and prognosis, as well as methods for determining the presence of one or more types of cancer.

[0004] Exemplary embodiments provide a method for determining the presence of at least one type of cancer. The method includes staining a portion of a synthetic tissue control (STC). The STC includes normal cells and cancer cells of a certain cancer type co-cultured based on at least one cell culture factor. The at least one co-culture factor includes the following factors: the type of cancer cells to be cultured, the ratio of cancer cells to be co-cultured with normal cells, the seeding concentration of the cultured cells, the type of cell growth supplement used to promote the co-culture of the cells, and the concentration of the cell growth supplement used to promote the co-culture of the cells. The method further includes observing the stained portion of the STC to determine the presence of one or more biomarker types, wherein the one or more biomarker types indicate the presence of cancer cells.

[0005] Exemplary embodiments provide a method for forming a synthetic tissue control for use in determining the presence of cancer, the method comprising culturing cells, including normal cells and cancer cells of a cancer type, based on at least one cell culture factor, the at least one cell culture factor including the type of cancer cells to be cultured, the ratio of cancer cells to normal cells to be cultured, and the seeding concentration of the cells to be cultured.

[0006] Another exemplary embodiment provides a synthetic tissue microarray. The synthetic tissue microarray includes a plurality of STCs, each of which includes normal cells and cancer cells of a cancer type. The normal cells and cancer cells are cultured based on at least one cell culture factor. The at least one cell culture factor includes the type of cancer cells to be cultured, the ratio of cancer cells to normal cells to be cultured, the seeding concentration of the normal cells and cancer cells to be cultured, and the type of cell growth supplement used to promote the culture and growth of the normal cells and cancer cells.

[0007] Further details of the disclosed embodiments are provided in the following detailed description and corresponding drawings.

[0008] Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, which are incorporated herein by reference. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram of a synthetic tissue microarray containing four controls, according to one embodiment. [Figure 2A] FIG. 1 shows a side view of a flowable gel that has solidified to form a cell retainer, according to one embodiment. [Figure 2B] 2B shows a side view of the cell holder of FIG. 2A with a cavity within the cell holder, according to one embodiment. [Figure 2C] 2B shows a top view of the cell holder of FIG. 2A, according to one embodiment. [Figure 2D] 2B shows another top view of the cell holder of FIG. 2A with dye added to co-cultured cells deposited within the cavity, according to one embodiment. [Figure 3A] 1 shows an image of a synthetic tissue containing breast cancer cells stained with a specific antibody against HER-2 / nue to show the presence of HER-2 / nue expression, according to one embodiment. [Figure 3B] 1 shows an image of breast tumor tissue stained with a specific antibody against HER-2 / nue to show the presence of HER-2 / nue expression, according to one embodiment. [Figure 3C] 3B shows an image of the composite tissue of FIG. 3A stained with pre-absorbed antibody HER-2 / nue as a test of the specificity of the staining, according to one embodiment. [Figure 3D] 3C shows an image of the breast tumor tissue of FIG. 3B stained with pre-absorbed antibody HER-2 / nue as a test of the specificity of the staining, according to one embodiment. [Figure 4A] 1 shows an image of a synthetic tissue stained to show the presence of the E-cadherin marker, according to one embodiment. [Figure 4B]1 shows an image of breast tumor tissue stained to show the presence of the E-cadherin marker, according to one embodiment. [Figure 4C] 1 shows an image of synthetic tissue stained to show the presence of estrogen receptor markers, according to one embodiment. [Figure 4D] 1 shows an image of breast tumor tissue stained to show the presence of estrogen receptor markers, according to one embodiment. [Figure 4E] 1 shows an image of synthetic tissue stained to show the presence of a marker of cell proliferation (Ki-67), according to one embodiment. [Figure 4F] 1 shows an image of breast tumor tissue stained to show the presence of a marker for cell proliferation (Ki-67), according to one embodiment. [Figure 5] 1 shows examples of antibodies for detecting different types of cancer covered by the synthetic tissue controls and synthetic tissue microarrays described herein, according to one embodiment.

[0010] The depicted diagrams are merely examples and are not intended to assert or imply any limitation with regard to the environment, architecture, design, or process in which different embodiments may be implemented. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description 3-D synthetic tissue controls (STCs) are generated by co-culturing normal cells and certain cancer cells in suspension under defined and controlled conditions. As defined herein, "normal" cells include non-tumor cells. Normal cells can be formed from stromal cells and other suitable cell types.

[0012] STCs reproducibly display the expected patterns and levels of tumor tissue-related cellular and extracellular (ECM) markers and architecture, closely resembling tumor tissue. Examples of the close resemblance between STCs and tumor tissue are shown in Figures 3A-3D and 4A-4E. 3-D synthetic tissue microarray controls (STMCs) are composed of multiple STCs. In a preferred embodiment, STCs and STMCs ​​are prepared as formalin-fixed, paraffin-embedded (FFPE) blocks or pre-cut into sections for various markers for use in pathology laboratories. Examples of compatible markers include the various markers detailed below and shown in the figures. STC and STMC blocks and sections can be used as both positive and negative controls for IHC and ISH staining of tumor tissue and other diseased tissues. The process of forming STCs and STMCs ​​and using them to detect the presence of cancer is described in detail in the following paragraphs.

[0013] Cell culture of STCs and STMCs

[0014] STCs are typically cultured in near-gravity culture in the form of formalin-fixed, paraffin-embedded (FFPE) cell blocks, with each STC containing certain cancer cells and stromal cells. In some embodiments, cancer cells and normal stromal cells are cultured separately in cell culture flasks. In one such embodiment, approximately 5 micrograms of DNase per milliliter of cell culture medium is added to each cell culture flask containing either cancer cells or normal stromal cells. The addition of DNase prevents clumping of cancer cells or normal stromal cells harvested from the cell culture flask. As a result, the cell count of cancer cells and / or normal stromal cells can be accurately determined, and non-clumped cancer cells and normal stromal cells can be harvested.

[0015] In some embodiments, two or more cell types (i.e., cancer cells and normal stromal cells) are co-cultured under precisely defined conditions and in a controlled environment. In some embodiments, cancer cells and stromal cells are co-cultured in multiple cell culture bags of cells. As defined herein, a cell culture bag is a container with built-in ports for adding and removing cells. In some embodiments, the cell culture bag is sterile. In further embodiments, the built-in ports of the cell culture bag include airtight caps. The bags hold the cancer cells and stromal cells during the production phase of the co-culture. In some embodiments, approximately 80 million stromal and / or cancer cells may be harvested from each cell culture bag after approximately 10 days of co-culture. In other embodiments, approximately 30 to 120 million stromal and / or cancer cells may be harvested from each cell culture bag after approximately 2 weeks of co-culture. The dimensions of the cell culture bags may vary based on the types of cells being co-cultured and the type of nutrients optimal for co-culturing such cells. In one such embodiment, approximately 5 micrograms of DNase per milliliter of cell culture medium is added to each cell culture bag containing cancer cells and normal stromal cells, and the cell culture bag is placed in a bioreactor in a CO2 incubator during the co-culture process. The addition of DNase facilitates maintaining the cells in a suspended, non-aggregated state during co-culture. More specifically, the addition of DNase promotes the maintenance of non-aggregated normal stromal cells, thereby enabling the normal stromal cells to form a homogenous core. Furthermore, the addition of DNase promotes the maintenance of non-aggregated cancer cells, facilitating the infiltration of the non-aggregated cancer cells into the homogenous core. In another such embodiment, approximately 1 microgram of fibronectin per milliliter of cell culture medium is added on the first day of co-culture to each cell culture bag containing cancer cells and normal stromal cells. In such an embodiment, the addition of fibronectin promotes the formation of a basement membrane-like structure during the early stages of co-culture cell growth. Furthermore, the addition of fibronectin promotes improved contact between the co-cultured cancer cells and normal stromal cells. The formation of basement membrane-like structures promotes the resemblance of co-cultured cells to actual tumor tissue.

[0016] In a preferred embodiment, cancer cells and stromal cells are co-cultured in a near-weightless environment for 8 to 12 days. The co-culture is further configured to maintain CO2 concentrations and temperatures inside and outside the chamber at levels based on at least one cell culture factor, allowing the cells to develop characteristics similar to or identical to those of actual tumor tissue. A motorized rotating device holds and slowly rotates the cell culture chamber at a speed based on at least one cell culture factor, allowing the cells to develop characteristics similar to or identical to those of actual tumor tissue.

[0017] Cell culture factors include, but are not limited to, the type of cancer cells cultured, the ratio of cancer cells to stromal cells, the seeding concentration of the cultured cells, and the concentration and type of cell growth supplements used. These factors promote cell culture to produce characteristics similar to or identical to those of actual tumor tissue. In one exemplary embodiment, the ratio of breast cancer cell line (MCF.7) cancer cells to normal stromal cells (fibroblasts) to produce STCs is 99.8 to 1, respectively. Additionally, 10 micrograms of insulin is used as a growth factor supplement to promote MCF7 proliferation. Furthermore, the seeding concentration of MCF.7 cancer cells is 18,750 cells per milliliter, while the seeding concentration of fibroblasts is 187,876 cells per milliliter. Similarly, STMCs ​​are cultured according to the above process. In one embodiment, STCs and STMCs ​​are cultured according to one of the above-identified factors. In another embodiment, STCs and STMCs ​​are cultured according to two of the above-identified factors. In a further embodiment, the STCs and STMCs ​​are cultured based on three of the above-identified factors. In a further embodiment, the STCs and STMCs ​​are cultured based on all of the above-identified factors.

[0018] STCs and STMCs ​​may be cultured based on at least one of the aforementioned cell culture factors to provide control "sham" tissues with known expression patterns and levels of various markers, including proteins, RNA, DNA, and other components of interest, for diagnosis, prognosis, and patient selection for specific / targeted therapies. STCs and STMCs ​​may be cultured for standardized expression of markers used in IHC as diagnostic and predictive markers for treatment response, exemplified by testing for epidermal growth factor receptor-2 (HER-2 / nue), estrogen receptor (ER), progesterone receptor (PR), Ki-67, and other types of appropriate diagnostic and predictive markers for treatment response. Furthermore, STCs and STMCs ​​may be cultured for standardized expression of markers used in IHC as predictive markers for treatment response, exemplified by testing for HER-2 / nue, Met4, and other types of appropriate predictive markers for treatment response. Furthermore, STCs and STMCs ​​may be cultured for standardized expression of markers used in immunofluorescence.

[0019] STCs and STMCs ​​may also be cultured to provide consistent expression levels when observed via fluorescent in situ hybridization (FISH)-based techniques for RNA and / or DNA markers. In certain embodiments, expression may include expression levels of RNA translocation. In other embodiments, expression may include expression levels of DNA mutations. STCs and STMCs ​​may also be cultured to provide consistent expression levels when observed via chromogenic in situ hybridization (CISH)-based techniques for RNA and / or DNA markers. In certain embodiments, expression may include expression levels of RNA translocation and / or DNA mutations. Thus, the choice of cell line used to generate STCs or STMCs ​​can provide a nearly limitless range of biomarkers.

[0020] Culturing STCs and STMCs ​​based on at least one of the aforementioned cell culture factors can consistently provide various expression levels of biomarkers for use in IHC and ISH staining. In some embodiments, STCs and STMCs ​​are cultured to provide highly expressed (HE or 3+) biomarkers for use in IHC and ISH staining. In other embodiments, STCs and STMCs ​​are cultured to have moderately expressed (ME or 2+) biomarkers for use in IHC and ISH staining. In further embodiments, STCs and STMCs ​​are cultured to have low expressed (LE or 1+) biomarkers for use in IHC and ISH staining. In some embodiments, co-cultured cells from multiple cell culture bags are settled into a centrifuge tube, the co-cultured cells are mixed, and the co-cultured cells are combined for processing and embedding. As described herein, the co-cultured cell product is tightly wrapped in biopsy filter paper and placed in a tissue processing device described herein, such as a centrifuge or another processing device.

[0021] Processing and embedding of STCs and STMCs

[0022] The cultured STCs are processed and then embedded. A cell holder with a cavity utilized to retain the co-cultured cells from at least two cell culture bags is utilized to retain the co-cultured cells during processing and embedding. In some embodiments, the cell holder is formed from a flowable gel that solidifies at room temperature. In some embodiments, once the flowable gel has solidified, it is placed in a reservoir having a shape similar to the desired shape of the flowable gel. An object is inserted into the flowable gel while it solidifies, and then the object is removed from the flowable gel after it has solidified to form a cavity. The size and shape of the cavity are determined by the external shape and outer dimensions of the object. In some embodiments, different objects with different outer sizes and shapes are utilized to form cavities of different sizes and shapes based on the desired amount of co-cultured cells to be processed, the type of cancer cells to be processed, the desired amount of STCs, and other factors described herein. The cavities securely hold the co-cultured cells and prevent them from dissipating into the surrounding medium during the processing and embedding process described herein.

[0023] As described herein, the cavity is operable to retain co-cultured cells recovered from two cell culture bags. In some embodiments, the cavity is operable to store co-cultured cells from additional cell culture bags, thereby further increasing the yield of desired cell cores. In some embodiments, a color dye is added to the recovered co-cultured cell product when it is transferred to the cavity of the cell holder. More specifically, a dye having a predetermined color can be added to the recovered co-cultured cell product without touching the product, thereby keeping the dye trapped within the cavity. Adding a color dye to the recovered co-cultured cell product before embedding it in paraffin facilitates an accurate means of identifying the recovered co-cultured cell product. In some embodiments, a filter medium such as biopsy filter paper is then wrapped around the cell holder containing the recovered co-cultured cell product and dye within its cavity. The cell holder is then placed in a histological cassette and transferred to a tissue processing tray. Additional processes, such as vacuum infiltration and paraffin embedding, may then be performed on the recovered co-cultured cell product as a block. In some embodiments, the recovered co-cultured cell product is fixed in formalin for subsequent processing and embedding. In other embodiments, the recovered co-cultured cell product is fixed in Bouin's solution or another fixative for subsequent processing and embedding. As described herein, the cavity is operable to retain co-cultured cells recovered from at least two cell culture bags. Additional description of the cell holder, the formation of the cell holder, and the deposition of co-cultured cells into the cavity of the cell holder is provided in the following paragraphs and is illustrated in at least Figures 2A-2D.

[0024] In one embodiment, the diameter of the cultured STCs is approximately 0.04 cm. In another embodiment, the diameter of the cultured STCs may be within the range of 0.01 to 0.04 cm. Unlike STCs, the diameter of the tissue specimen may be 2 to 4 cm. Considering the size of the cultured STCs, a processing and embedding device with a mesh with a pore size of approximately 0.001 cm is used to hold the tissue specimen during the embedding process. In another embodiment, the pore size of the embedding device is within the range of 0.001 to 0.004 cm. Multiple cultured STCs that provide the desired expression levels of the desired biomarkers are embedded to form an STMC.

[0025] Staining of STCs and STMCs

[0026] Sections from each block of embedded STCs are evaluated using IHC staining techniques to identify individual constructs. In some embodiments, the individual constructs within the embedded STCs represent 50-60% of a total of approximately 500 individual constructs, with the desired combination of cancer cells and normal stromal cells, and infiltration of a normal stromal cell core by cancer cells. In other embodiments, the individual constructs within the embedded STCs represent 80% of a total of 500-600 individual constructs, with the desired combination of cancer cells and normal stromal cells, and infiltration of a normal stromal cell core by cancer cells. In some embodiments, only constructs with characteristics similar or identical to those of actual tumor tissue are selected as STCs.

[0027] Individual constructs from each block containing specific combinations of co-cultured cell types with characteristics similar or identical to those of actual tumor tissue are mechanically removed from the original block and used to construct STMCs. STMCs ​​are constructed to contain multiple types of cancer cells with various expression levels and patterns of markers of interest. Laboratory operators may view the STCs and STMCs ​​through various devices, such as microscopes, whole slide imaging (WSI) devices, and other suitable devices for observing biomarker expression.

[0028] Figure 1 is a diagram of a synthetic tissue microarray 100 including four STCs 101, 102, 103, and 104, according to one embodiment. In the embodiment shown in Figure 1, the controls 101, 102, 103, and 104 are placed adjacent to a test tissue 105 on the same histological slide. The controls 101, 102, 103, and 104 and the test tissue 105 are stained with one or more stains for different types of biomarkers.

[0029] In some embodiments, the synthetic tissue microarray 100 is stained to monitor the expression of markers used in IHC as diagnostic and predictive markers of treatment response. In other embodiments, the synthetic tissue microarray 100 is stained to monitor the expression of markers used in IHC as predictive markers of treatment response. In a further embodiment, the synthetic tissue microarray 100 is stained and monitored for expression levels of RNA and DNA markers by FISH techniques. In a further embodiment, the synthetic tissue microarray 100 is stained and monitored for expression levels of RNA and DNA markers by CISH techniques.

[0030] In some embodiments, the synthetic tissue microarray 100 provides positive controls for at least one type of cancer. In other embodiments, some controls on the synthetic tissue microarray 100 provide positive controls, and other controls on the synthetic tissue microarray 100 provide negative controls. The synthetic tissue microarray 100 may be cultured to provide high, moderate, or low expression of markers. The embodiment shown in FIG. 1 includes four controls 101, 102, 103, and 104, although the synthetic tissue microarray 100 may be formed from a different number of controls. Laboratory operators may examine the synthetic tissue microarray 100 under various equipment, such as microscopes and WSI devices, and compare the stained controls to stained test tissue to determine the presence or absence of marker expression in the test tumor tissue.

[0031] FIG. 2A shows a side view of a fluid gel 201 solidifying to form a cell retainer 200, according to one embodiment. The fluid gel 201 is deposited adjacent to a cooling object 220, and the temperature of the fluid gel 201 is cooled, thereby solidifying the fluid gel 201 to form the cell retainer 200. In some embodiments, the fluid gel 201 is solid at room temperature and is first heated so that the fluid gel 201 becomes fluid. In some embodiments, the fluid gel 201 is placed within a reservoir during the solidification process, and assumes a shape approximately defined by the interior surface of the reservoir. In some embodiments, the cooling object 220 is ice, which facilitates the cooling of the fluid gel 201. An object 210 is inserted into the fluid gel 201 while the fluid gel 201 is solidifying, and is removed from the fluid gel 201 after the fluid gel 201 has solidified to form the cavity of the cell retainer 200 (shown in FIGS. 2B-2D). In some embodiments, the object 201 is a tube having a shape and dimensions approximating the desired shape and dimensions of the cavity. As described herein, the cavity is operable to hold co-cultured cells from at least two cell culture bags described herein.

[0032] FIG. 2B shows a side view of the cell holder 200 of FIG. 2A having a cavity 202 therein, according to one embodiment. FIG. 2C shows another image of the cell holder 200 of FIG. 2A having a cavity 202, according to one embodiment. Although FIGS. 2B and 2C show one cavity 202 formed in the cell holder 200, multiple cavities may be formed in the cell holder 200, each operatively holding co-cultured cells from at least two cell culture bags. Furthermore, the cavity in FIG. 2C penetrates opposing surfaces of the cell holder 200, while other cavities may not penetrate the cell holder 200. As described herein, the cavity 202 may take on a variety of shapes and sizes, including the shapes shown in FIGS. 2B and 2C.

[0033] FIG. 2D shows another top view of the cell retainer 200 of FIG. 2A with a dye added to the co-cultured cells 204 deposited in the cavity 202, according to one embodiment. In the embodiment of FIG. 2D, a blue dye is added to the co-cultured cells 204 deposited in the cavity 202. The amount of dye added can be based on various factors, including the number of co-cultured cells deposited in the cavity 202, the type of cancer cells deposited in the cavity 202, the size and dimensions of the cavity 202, and other factors described herein. In some embodiments, a pre-selected colored dye is carefully added to the co-cultured cell product in the cavity 202 to ensure that the colored dye remains confined within the cavity 202. While FIG. 2D shows blue dye added to the cavity 202, a different colored dye may be added to facilitate identification of the co-cultured cells. In some embodiments, a filter medium, such as filter paper, is added above the cavity. In some embodiments, the cell retainer 200 is then transferred to a pathology cassette (not shown), and the pathology cassette is processed to form an STC. In some embodiments, the STC is then embedded in a paraffin block to form an STMA.

[0034] Figure 3A shows an image of synthetic tissue containing breast cancer cells according to one embodiment. Figure 3B shows an image of breast tumor tissue according to one embodiment. The synthetic tissue shown in Figure 3A was cultured under conditions described herein. As shown in Figures 3A and 3B, the synthetic tissue containing breast cancer cells and actual breast tumor tissue exhibit very similar characteristics.

[0035] Figure 3C shows an image of the synthetic tissue of Figure 3A after staining the synthetic tissue with a preabsorbed antibody against HER-2 / nue, according to one embodiment, demonstrating the specificity of the staining for the HER-2 / nue marker. Figure 3D shows an image of the breast tumor tissue of Figure 3B after staining the tumor tissue with a preabsorbed antibody against HER-2 / nue, according to one embodiment, demonstrating the specificity of the staining for the HER-2 / nue marker. As shown in Figures 3C and 3D, the stained synthetic tissue and the stained tumor tissue exhibit very similar characteristics, allowing the synthetic tissue to be used as a control for the standard expression of markers used in IHC as diagnostic or predictive markers of treatment response, such as HER-2 / nue. Other examples of marker expression for use in IHC as predictive markers of treatment response include Met4 and other suitable diagnostic or predictive markers of treatment response. In further embodiments, the synthetic tissue shown in Figures 3A and 3C can also provide expression levels of RNA and DNA markers when observed via FISH techniques. In a further embodiment, the synthetic tissues shown in Figures 3A and 3C may also provide expression levels of RNA and DNA markers when viewed via CISH techniques.

[0036] Figure 4A shows an image of synthetic tissue stained to show the presence of the E-cadherin marker, according to one embodiment. Figure 4B shows an image of breast tumor tissue stained to show the presence of the E-cadherin marker, according to one embodiment. Figure 4C shows an image of synthetic tissue stained to show the presence of an estrogen receptor marker, according to one embodiment. Figure 4D shows an image of breast tumor tissue stained to show the presence of an estrogen receptor marker, according to one embodiment. Figure 4E shows an image of synthetic tissue stained to show the presence of a proliferation (Ki-67) marker, according to one embodiment. Figure 4F shows an image of breast tumor tissue stained to show the presence of a proliferation (Ki-67) marker, according to one embodiment.

[0037] The synthetic tissue shown in Figures 4A, 4C, and 4E was cultured under the conditions described herein. As shown in Figures 4A-4F, the synthetic tissue and stained tumor tissue exhibited highly similar characteristics, allowing the synthetic tissue to be used in IHC as a diagnostic and predictive marker of treatment response, e.g., testing for E-cadherin, ER, progesterone receptor, and Ki-67, as well as other suitable types of diagnostic and predictive markers of treatment response. In other embodiments, the synthetic tissue shown in Figures 4A, 4C, and 4E may be used to provide expression of biomarkers for use in IHC as predictive markers of treatment response. In further embodiments, the synthetic tissue shown in Figures 4A, 4C, and 4E may also provide expression levels of RNA and DNA markers when viewed via FISH techniques. In further embodiments, the synthetic tissue shown in Figures 4A, 4C, and 4E may also provide expression levels of RNA and DNA markers when viewed via CISH techniques.

[0038] Figure 5 shows examples of antibodies for detecting different types of cancer covered by the synthetic tissue controls and synthetic tissue microarray controls described herein, according to one embodiment. As shown in Figure 5, STCs and STMCs ​​can be used to test for various types of cancer, including, but not limited to, breast cancer, lung cancer, liver cancer, thyroid cancer, prostate cancer, colon cancer, cervical cancer, kidney cancer, ovarian cancer, melanoma cancer, brain cancer, leukemia, lymphoma, and other types of cancer.

[0039] The above disclosed embodiments are presented for purposes of illustration and to enable those skilled in the art to practice the disclosed embodiments, but are not intended to be exhaustive or to be limited to the disclosed forms. Many insubstantial modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. For example, while the flowcharts show a serial process, some steps / blocks may be performed in parallel or out of sequence, or combined into a single step / block. The claims are intended to broadly cover the disclosed embodiments and any such modifications. Furthermore, the following clauses represent additional embodiments of the present disclosure and should be considered within the scope of the present disclosure:

[0040] Clause 1. A method for increasing the yield of co-cultured cells in a synthetic tissue control, the method comprising: solidifying a fluid gel to form a solid cell holder; forming a cavity in the cell holder to hold co-cultured cells, the cavity operable to hold a plurality of cell culture bags of co-cultured cells, the co-cultured cells comprising normal cells and cancer cells of a certain cancer type co-cultured based on at least one cell culture factor, the at least one co-culture factor comprising the type of cancer cells to be co-cultured, the ratio of the cancer cells to the normal cells to be co-cultured, the seeding concentration of the normal cells and the cancer cells to be co-cultured, a cell growth supplement used to promote the co-culture of the normal cells and the cancer cells, and the concentration of the cell growth supplement used to promote the co-culture of the normal cells and the cancer cells; the method also comprising depositing co-cultured cells derived from at least two of the plurality of cell culture bags of co-cultured cells into the cavity of the cell holder; and treating the co-cultured cells to form the synthetic tissue control.

[0041] Clause 2: The method of clause 1, further comprising adding a certain amount of dye to the cavity during the processing of the co-cultured cells, and identifying the co-cultured cells deposited in the cavity.

[0042] Clause 3: The method of clause 1 or 2, further comprising determining the amount of dye to add to the cavity based on the number of co-cultured cells deposited in the cavity.

[0043] Clause 4. The method of at least one of clauses 1 to 3, wherein forming the cavity in the cell support comprises: inserting an object into the flowable gel while the flowable gel is solidifying, a portion of the object having a shape that defines the cavity; and removing the object after the flowable gel has solidified to form the cavity.

[0044] Clause 5. The method of at least one of clauses 1-4, further comprising embedding said synthetic tissue control in a paraffin block to form a synthetic tissue microarray.

[0045] Clause 6. The method of at least one of clauses 1-5, further comprising: adding a filter medium over the cavity; and transferring the cell retainer to a pathology cassette and processing the pathology cassette to form the synthetic tissue control.

[0046] Clause 7: A method for forming a synthetic tissue control for use in determining the presence of cancer, the method comprising: co-culturing cell culture bags of a plurality of cells containing normal cells and cancer cells of a certain cancer type based on at least one cell culture factor, the at least one cell culture factor comprising the type of the cancer cells to be cultured, the ratio of the cancer cells to the normal cells to be cultured, and the seeding concentration of the normal cells and the cancer cells to be cultured; the method also comprising depositing cell culture bags of at least two co-cultured cells among the plurality of cell culture bags of co-cultured cells into a cavity of a cell holder; treating the co-cultured cells from the cell culture bags of at least two co-cultured cells deposited in the cavity; adding a certain amount of dye to the cavity and identifying the co-cultured cells deposited in the cavity during the treatment of the co-cultured cells; and forming the synthetic tissue control from the co-cultured cells deposited in the cavity.

[0047] Clause 8. The method of clause 7, wherein co-culturing cell culture bags of the plurality of cells comprises co-culturing the normal cells and the cancer cells in a cell culture chamber configured to maintain a CO2 concentration and a temperature in the cell culture chamber at levels based on the at least one cell culture factor.

[0048] Clause 9. The method of clause 7 or 8, wherein co-culturing the cell culture bags of the plurality of cells further comprises maintaining the cell culture chamber in an electric rotating device operable to: hold the synthetic tissue control; and rotate the cell culture chamber at a rate based on the at least one cell culture factor.

[0049] Clause 10. The method of at least one of clauses 7 to 9, wherein co-culturing the cell culture bag of the plurality of cells further comprises culturing the co-cultured cells to provide expression of markers for use in IHC as diagnostic and predictive markers of therapeutic response.

[0050] Clause 11. The method of at least one of clauses 7 to 9, wherein co-culturing cell culture bags of the plurality of cells further comprises culturing the co-cultured cells to provide expression of a marker for use in IHC as a diagnostic or predictive marker of therapeutic response.

[0051] Clause 12: The method of at least one of clauses 7 to 9, wherein co-culturing the cell culture bags of the plurality of cells further comprises culturing the co-cultured cells to provide consistent expression levels of RNA markers and DNA markers when observed by FISH technology.

[0052] Clause 13: The method of at least one of clauses 7 to 9, wherein co-culturing cell culture bags of the plurality of cells further comprises culturing the co-cultured cells to provide consistent expression levels of RNA markers and DNA markers when observed by CISH technology.

[0053] Clause 14: The method of at least one of clauses 7 to 13, further comprising adding DNase to promote disaggregation of said normal cells and said cancer cells.

[0054] Clause 15. The method of at least one of clauses 7 to 14, further comprising: forming a homogenous core having non-aggregated normal cells; and infiltrating the non-aggregated cancer cells into the homogenous core.

[0055] Clause 16: The method of at least one of clauses 7 to 15, further comprising adding fibronectin to promote disaggregation of said normal cells and said cancer cells.

[0056] Clause 17. A synthetic tissue microarray comprising a plurality of synthetic tissue controls, each synthetic tissue control of the plurality of synthetic tissue controls comprising: normal cells; and cancer cells of a cancer type, wherein the normal cells and the cancer cells are co-cultured in at least two cell culture bags among a plurality of cell culture bags of co-cultured normal cells and cancer cells, and wherein the plurality of cell culture bags of co-cultured normal cells and the cancer cells are co-cultured based on at least one cell culture factor, including the type of cancer cells to be cultured, the ratio of the cancer cells to the normal cells to be cultured, the seeding concentration of the normal cells and the cancer cells to be cultured, and the type of cell growth supplement used to promote the culture of the normal cells and the cancer cells.

[0057] Clause 18. The synthetic tissue microarray of clause 17, wherein said normal and cancer cells are co-cultured to provide high expression of markers for use in IHC or ISH staining.

[0058] Clause 19. The synthetic tissue microarray of clause 17 or 18, wherein the normal and cancer cells are co-cultured to provide moderate expression of markers for use in IHC or ISH staining.

[0059] Clause 20. The synthetic tissue microarray of at least one of clauses 17 to 19, wherein the normal cells and cancer cells are co-cultured to provide low expression of markers for use in IHC or ISH staining.

[0060] As used herein, "near zero gravity environment" is defined to include a zero gravity environment.

[0061] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, as used in this specification and / or claims, the terms "comprise" and / or "comprising" specify the presence of stated features, steps, operations, elements, and / or components, but are understood not to exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. Furthermore, the steps and components described in the above embodiments and figures are merely exemplary and are not intended to imply that any particular step or component is a requirement of a claimed embodiment.

Claims

1. 1. A method for forming a synthetic tissue control for use in determining the presence of cancer, said method comprising: co-culturing a plurality of cells in each of at least two cell culture bags, the plurality of cells comprising normal cells and cancer cells of a cancer type based on at least one cell culture factor, the at least one cell culture factor comprising a type of the cancer cells to be cultured, a ratio of the cancer cells to the normal cells to be cultured, and a seeding concentration of the normal cells and the cancer cells to be cultured; the method also comprising: depositing the co-cultured cells of the at least two cell culture bags into the cavities of the cell holder in which the flowable gel has solidified; configured to add the co-cultured cells from the at least two cell culture bags deposited in the cavity and retain the plurality of cells from each of the at least two cell culture bags in the cavity during the addition of the co-cultured cells; After adding the co-cultured cells, adding a certain amount of dye to the cavity to identify the co-cultured cells deposited in the cavity during the addition of the co-cultured cells; and The method of forming the synthetic tissue control from the co-cultured cells deposited in the cavity, the cell culture bag being a container with built-in ports for adding and removing cells.

2. co-culturing the plurality of cells in the cell culture bag further comprises co-culturing the normal cells and the cancer cells in the cell culture bag in a cell culture chamber; The cell culture chamber is configured to: ２ The method of claim 1 , configured to maintain concentrations and temperatures at levels based on the at least one cell culture factor.

3. Co-culturing the plurality of cells in the cell culture bag comprises: Retaining said synthetic tissue control; and 3. The method of claim 2, further comprising maintaining the cell culture chamber in a motorized rotating device operable to rotate the cell culture chamber at a rate based on the at least one cell culture factor.

4. 3. The method of claim 2, wherein co-culturing the plurality of cells in the cell culture bag further comprises culturing the co-cultured cells to provide expression of markers for use in immunohistochemistry (IHC) as diagnostic and predictive markers of therapeutic response.

5. 3. The method of claim 2, wherein co-culturing the plurality of cells in the cell culture bag further comprises culturing the co-cultured cells to provide expression of a marker for use in immunohistochemistry (IHC) as a diagnostic or predictive marker of therapeutic response.

6. 3. The method of claim 2, wherein co-culturing the plurality of cells in the cell culture bag further comprises culturing the co-cultured cells to provide consistent expression levels of RNA and DNA markers when observed by fluorescence in situ hybridization (FISH) techniques.

7. 3. The method of claim 2, wherein co-culturing the plurality of cells in the cell culture bag further comprises culturing the co-cultured cells to provide consistent expression levels of RNA and DNA markers when observed with chromogenic in situ hybridization (CISH) techniques.

8. 10. The method of claim 1, further comprising adding DNase to promote disaggregation of the normal and cancer cells.

9. forming a homogenous core with non-aggregated normal cells; and 9. The method of claim 8, further comprising infiltrating the homogenous core with non-aggregated cells of the cancer cells.

10. 10. The method of claim 1, further comprising adding fibronectin to promote disaggregation of the normal and cancer cells.

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