Cell yield for synthetic tissue control and synthetic tissue microarray control

Abstract

To provide a methods for forming synthetic tissue controls and synthetic tissue microarray controls for IHC and ISH tests for cancer diagnosis and prognosis as well as a method for determining presence of one or more types of cancer.SOLUTION: Provided is a method of increasing the yield of co-cultured cells of a synthetic tissue control. The method comprises the following: solidifying a fluid gel to form a solid cell support; and forming a cavity in the cell support to retain the co-cultured cells, in which the co-cultured cells include normal cells and cancer cells of a certain cancer type and at least one co-culture factor includes the type of the cancer cells co-cultured, the ratio of the cancer cells to the normal cells co-cultured. The method also comprises the following: depositing, in the cavity of the cell support, co-cultured cells from at least two of a plurality of cell culture bags of the co-cultured cells; and treating the co-cultured cells to form the synthetic tissue control.SELECTED DRAWING: 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 reproducibility of test results by 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 very limited quantities, and when such controls 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 types of controls do not show consistent patterns and levels of cellular expression of a given marker, or the heterogeneity of said expression that is ubiquitous in tumor tissues. Therefore, 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. Summary of the Invention

[0003] The disclosed embodiments provide methods for forming synthetic tissue controls and synthetic tissue microarray controls for IHC and ISH testing 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 cancer type co-cultured based on at least one cell culture factor. The at least one co-culture factor includes the following factors: a type of cancer cells to be cultured, a ratio of cancer cells to be co-cultured with normal cells, a seeding concentration of the cells to be cultured, a type of cell growth supplement used to promote the co-culture of the cells, and a concentration of a 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, where the one or more biomarker types indicate the presence of the 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] According to another exemplary embodiment, a synthetic tissue microarray is provided. The synthetic tissue microarray includes a plurality of STCs, each STC of the plurality of STCs includes normal cells and cancer cells of a cancer type. The normal cells and the cancer cells are cultured based on at least one cell culture factor. The at least one cell culture factor includes a type of cancer cells to be cultured, a ratio of cancer cells to normal cells to be cultured, a seeding concentration of the normal cells and the cancer cells to be cultured, and a type of cell growth supplement to be used to promote the culture and growth of the normal cells and the 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 description of the drawings]

[0009] [Figure 1] FIG. 2 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] FIG. 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] FIG. 2B shows a top view of the cell holder of FIG. 2A, according to one embodiment. [Figure 2D] FIG. 2B shows another top view of the cell holder of FIG. 2A with dye added to the 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 preabsorbed 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 preabsorbed 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 a synthetic tissue stained to show the presence of an estrogen receptor marker, 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 for 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. [Diagram 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 limitation with respect to the environment, architecture, design, or process in which different embodiments may be implemented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 associated cellular and extracellular (ECM) markers and architecture that are highly similar to tumor tissue. Examples of the close similarity 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 and paraffin-embedded (FFPE) blocks or pre-cut into sections for various markers for use in pathology laboratories. Examples of compatible markers include various markers detailed in the following description 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 and other diseased tissues. The process of forming STCs and STMCs ​​and using STCs and STMCs ​​to detect the presence of cancer is detailed in the following paragraphs.

[0013] Cell culture of STCs and STMCs

[0014] STCs are typically grown in near-gravity culture in the form of formalin-fixed and paraffin-embedded (FFPE) cell blocks, with each STC containing certain cancer cells and stromal cells. In some embodiments, the 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 the cancer cells or the normal stromal cells. The addition of DNase prevents the aggregation of the cancer cells or normal stromal cells harvested from the cell culture flask. As a result, the cell count of the cancer cells and / or normal stromal cells can be accurately determined, and non-aggregated 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, the 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 an airtight cap. The bag holds 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 bag may vary based on the type of cells to be co-cultured and the type of nutrients that are 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 the cancer cells and normal stromal cells, and the cell culture bags are heated to 40° C. during the co-culture process. 2 The bioreactor is placed in an incubator. The addition of DNase helps the cells to remain suspended and non-aggregated during co-culture. More specifically, the addition of DNase helps the normal stromal cells to remain non-aggregated, thus allowing the normal stromal cells to form a homogenous core. Furthermore, the addition of DNase helps the cancer cells to remain non-aggregated, allowing the non-aggregated cancer cells to invade 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 in each cell culture bag containing the cancer cells and the 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 growth of the co-cultured cells. Furthermore, the addition of fibronectin promotes improved contact between the co-cultured cancer cells and the normal stromal cells. The formation of a basement membrane-like structure promotes the co-cultured cells to resemble actual tumor tissue.

[0016] In a preferred embodiment, the cancer cells and stromal cells are co-cultured in a near-gravity environment for 8-12 days. Additionally, CO 2 The cancer cells and stromal cells are co-cultured in a cell culture chamber configured to maintain a concentration and temperature at a level based on at least one cell culture factor to induce the cells to develop characteristics similar or identical to those of actual tumor tissue. A motorized rotation device holds and slowly rotates the cell culture chamber at a rate based on the at least one cell culture factor to induce the cells to develop characteristics similar or identical to those of actual tumor tissue.

[0017] The cell culture factors include, but are not limited to, the type of cancer cells to be cultured, the ratio of cancer cells to stromal cells, the seeding concentration of the cells to be cultured, and the concentration and type of cell growth supplements used. Facilitate the culture of cells to produce characteristics similar or identical to that of real tumor tissue. In an exemplary embodiment, the ratio of breast cancer cell line (MCF.7) cancer cells to normal stromal cells (fibroblasts) is 99.8 to 1, respectively, to generate STCs. In addition, 10 micrograms of insulin is used as a growth factor supplement to promote the proliferation of MCF7. 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 also cultured according to the above process. In one embodiment, STCs and STMCs ​​are cultured according to one of the factors identified above. In another embodiment, STCs and STMCs ​​are cultured according to two of the factors identified above. In a further embodiment, the STCs and STMCs ​​are cultured based on three of the factors identified above. In a further embodiment, the STCs and STMCs ​​are cultured based on all of the factors identified above.

[0018] STCs and STMCs ​​are cultured based on at least one of the aforementioned cell culture factors to provide control "sham" tissues with various markers of known expression patterns and expression levels, including proteins, RNA, DNA and other components of interest, for diagnosis, prognosis and patient selection for a particular specific / targeted therapy. STCs and STMCs ​​may be cultured for standard 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 suitable diagnostic and predictive markers for treatment response. Additionally, STCs and STMCs ​​may be cultured for standard expression of markers used in IHC as predictive markers for treatment response, exemplified by testing for HER-2 / nue, Met4, and other types of suitable predictive markers for treatment response. Additionally, STCs and STMCs ​​may be cultured for standard 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 ​​may provide an almost 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 high expression (HE or 3+) biomarkers for use in IHC and ISH staining. In other embodiments, STCs and STMCs ​​are cultured to have moderate expression (ME or 2+) biomarkers for use in IHC and ISH staining. In further embodiments, STCs and STMCs ​​are cultured to have low expression (LE or 1+) biomarkers for use in IHC and ISH staining. In some embodiments, co-cultured cells from multiple cell culture bags are precipitated into a centrifuge tube, the co-cultured cells are mixed, and the co-cultured cells are combined for processing and embedding. The co-cultured cell product is tightly wrapped with a 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 STC and STMC

[0022] The cultured STCs are treated and then embedded. A cell holder with a cavity utilized to hold the co-cultured cells from at least two cell culture bags is utilized to hold the co-cultured cells during the treatment and embedding of the co-cultured cells. In some embodiments, the cell holder is formed from a flowable gel that solidifies at room temperature. In some embodiments, once the flowable gel is solidified, the flowable gel 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 the flowable gel is solidifying, and then the object is removed from the flowable gel after the flowable gel has solidified to form a cavity. The size and shape of the cavity is defined by the external shape and external size of the object. In some embodiments, different objects with different external sizes and shapes are utilized to form cavities with different sizes and shapes based on the desired amount of co-cultured cells to be treated, the type of cancer cells to be treated, the amount of STCs desired, and other factors described herein. The cavities hold the co-cultured cells securely and prevent the co-cultured cells from dissipating into the surrounding medium during the treatment and embedding process described herein.

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

[0024] In one embodiment, the diameter of the cultured STC is approximately 0.04 cm. In another embodiment, the diameter of the cultured STC may be in the range of 0.01-0.04 cm. Unlike the STC, the diameter of the tissue specimen may be 2-4 cm. Considering the size of the cultured STC, a processing and embedding device having 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 in the range of 0.001-0.004 cm. A plurality of cultured STCs that provide the desired expression level of the desired biomarker are embedded to form an STMC.

[0025] Staining of STCs and STMCs

[0026] Sections from each block of embedded STC are evaluated by IHC staining techniques to identify individual constructs. In some embodiments, the individual constructs within the embedded STC represent 50-60% of the 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 STC represent 80% of the 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 similar or identical characteristics to actual tumor tissue are selected as STCs.

[0027] Individual constructs from each block containing a specific combination of co-cultured cell types with similar or identical characteristics to the 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 to observe the expression of biomarkers.

[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 further embodiments, the synthetic tissue microarray 100 is stained and monitored for expression levels of RNA and DNA markers by FISH techniques. In further embodiments, 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 of the synthetic tissue microarray 100 provide positive controls and other controls of the synthetic tissue microarray 100 provide negative controls. The synthetic tissue microarray 100 may be cultured to provide high, medium, or low expression of the marker. The embodiment shown in FIG. 1 includes four controls 101, 102, 103, and 104, but the synthetic tissue microarray 100 may be formed from a different number of controls. A laboratory operator may examine the synthetic tissue microarray 100 under various equipment, such as a microscope and WSI equipment, and compare the stained controls to stained test tissue to determine the presence or absence of expression of the marker in the test tumor tissue.

[0031] FIG. 2A shows a side view of the fluid gel 201 that is solidified to form the cell retainer 200, according to one embodiment. The fluid gel 201 is deposited proximate 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 is in a fluid state. In some embodiments, the fluid gel 201 is placed in 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. The 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 (as shown in FIGS. 2B-2D). In some embodiments, the object 201 is a tube having a shape and dimensions that approximate 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 as described herein.

[0032] FIG. 2B shows a side view of the cell holder 200 of FIG. 2A with a cavity 202 in the cell holder 200, according to one embodiment. FIG. 2C shows another image of the cell holder 200 of FIG. 2A with 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 the 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 various shapes and dimensions, 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 dye added to the co-cultured cells 204 deposited in the cavity, 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 may 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 trapped within the cavity 202. Although FIG. 2D shows a blue dye added to the cavity 202, a different colored dye may be added to help identify 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 a synthetic tissue containing breast cancer cells according to one embodiment. Figure 3B shows an image of a 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 the actual breast tumor tissue show very similar characteristics.

[0035] FIG. 3C shows an image of the synthetic tissue of FIG. 3A after staining the synthetic tissue with a preabsorbed antibody against HER-2 / nue, according to one embodiment, showing the specificity of the staining for the HER-2 / nue marker. FIG. 3D shows an image of the breast tumor tissue of FIG. 3B after staining the tumor tissue with a preabsorbed antibody against HER-2 / nue, according to one embodiment, showing the specificity of the staining for the HER-2 / nue marker. As shown in FIGS. 3C and 3D, the stained synthetic tissue and the stained tumor tissue show very similar characteristics, so the synthetic tissue can be used as a control for standard expression of markers used in IHC as diagnostic or predictive markers of treatment response, such as HER-2 / nue. Other examples of expression of markers used in IHC as predictive markers of treatment response include Met4 and other suitable diagnostic or predictive markers of treatment response. In a further embodiment, the synthetic tissue shown in FIGS. 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 observed via CISH technology.

[0036] FIG. 4A shows an image of synthetic tissue stained to show the presence of an E-cadherin marker, according to one embodiment. FIG. 4B shows an image of breast tumor tissue stained to show the presence of an E-cadherin marker, according to one embodiment. FIG. 4C shows an image of synthetic tissue stained to show the presence of an estrogen receptor marker, according to one embodiment. FIG. 4D shows an image of breast tumor tissue stained to show the presence of an estrogen receptor marker, according to one embodiment. FIG. 4E shows an image of synthetic tissue stained to show the presence of a proliferation (Ki-67) marker, according to one embodiment. FIG. 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 the stained tumor tissue show very similar characteristics, which allows the synthetic tissue to be used in IHC as a diagnostic and predictive marker of treatment response, for example, 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 cancers 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 cancers, 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 one 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, although the flow charts 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. In addition, 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 of 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 the co-cultured cells, the cavity being 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 cancer type co-cultured based on at least one cell culture factor, the at least one co-culture factor comprising a type of the cancer cells to be co-cultured, a ratio of the cancer cells to the normal cells to be co-cultured, a 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 a 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 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 quantity of a dye to the cavity during the processing of the co-cultured cells, and identifying the co-cultured cells deposited within the cavity.

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

[0043] Clause 4. The method of at least one of clauses 1-3, wherein forming the cavity in the cell retainer 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 controls in paraffin blocks 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, 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 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 comprises depositing cell culture bags of at least two co-cultured cells among the cell culture bags of the plurality of co-cultured cells into a cavity of a cell holder; treating the co-cultured cells from the cell culture bags of the at least two co-cultured cells deposited in the cavity; adding a certain amount of dye into 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, wherein the co-culturing of the cell culture bag of the plurality of cells comprises: 2 8. The method of claim 7, comprising co-culturing the normal cells and the cancer cells in the cell culture chamber configured to maintain a concentration and temperature at a level 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 bag of the plurality of cells further comprises maintaining the cell culture chamber within a motorized 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 a 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 a cell culture bag 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 according to 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 and DNA markers when observed by FISH technology.

[0052] Clause 13. The method according to 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 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 7-14, further comprising: forming a homogenous core having non-aggregated normal cells; and infiltrating said homogenous core with non-aggregated cancer cells.

[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 of a plurality of cell culture bags of co-cultured normal cells and cancer cells, and 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, comprising: a type of the cancer cells to be cultured, a ratio of the cancer cells to the normal cells to be cultured, a seeding concentration of the normal cells and the cancer cells to be cultured, and a 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 a marker for use in IHC or ISH staining.

[0058] Clause 19. The synthetic tissue microarray of clause 17 or 18, wherein said 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 a marker 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. Synthetic tissue controls and synthetic tissue microarray controls, Contains normal cells and cancer cells, and has morphological characteristics and marker expression patterns similar to tumor tissue, The normal cells and the cancer cells are co-cultured based on at least one cell culture factor; and the at least one cell culture factor (a) the type of cancer cells to be co-cultured; (b) the ratio of cancer cells to normal cells; (c) seeding concentration; (d) the type of cell growth supplement; and (e) Concentration of cell proliferation supplement A synthetic tissue control and a synthetic tissue microarray control selected from:

2. 2. The synthetic tissue control and synthetic tissue microarray control of claim 1, wherein the synthetic tissue microarray control comprises a plurality of synthetic tissue controls, each synthetic tissue control of the plurality of synthetic tissue controls having a different type of cancer cell or a different biomarker expression level.

3. 2. The synthetic tissue control and synthetic tissue microarray control of claim 1, The synthetic tissue control and the synthetic tissue microarray control are formed using a cell holder having a cavity for holding co-cultured cells from at least two cell culture bags.

4. 4. The synthetic tissue control and synthetic tissue microarray control of claim 3, wherein the cavity is formed by removing an object inserted into a fluid gel that solidifies at room temperature while the fluid gel is solidifying.

5. 4. The synthetic tissue control and synthetic tissue microarray control of claim 3, wherein the size and shape of the cavity is defined by the external shape and outer dimensions of the object.

6. The synthetic tissue control and synthetic tissue microarray control of claim 3, wherein the co-culture conditions are adjusted to achieve a desired cell core yield by retaining the co-cultured cells recovered from the at least two cell culture bags within the cavities of the cell holder.

7. 10. The synthetic tissue control and synthetic tissue microarray control of claim 1, wherein the cell growth supplement comprises insulin or fibronectin.

8. 2. The synthetic tissue control and synthetic tissue microarray control of claim 1, wherein the co-culture is performed in a substantially weightless environment.

9. 2. The synthetic tissue control and synthetic tissue microarray control of claim 1, wherein the cancer cells are selected from 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.

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