# US 10,501,717 B2 · Method and apparatus for isolating invasive and metastatic cells for evaluating therapeutics and prediction of metastatic capacity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,717 B2 - Document ID: US10501717B2 - Status: Granted - Kind: B2 - Application number: 15/112,408 - Filed: 2015-01-21 - Published: 2016-11-24 - Granted: 2019-12-10 - Earliest claimed: 2014-01-22 - Assignees: zPredicta, Inc. - Applicants: zPredicta, Inc. - Inventors: Julia Kirshner; Mukti Rajen Parikh - Web page: https://patentgrep.com/patent/US10501717B2 ## CPC - C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability} - A61P 35/00: Antineoplastic agents - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12M 25/04: {in combination with well or multiwell plates, i.e. culture inserts} - C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)} - C12M 35/08: {Chemical, biochemical or biological means, e.g. plasma jet, co-culture} - C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} - G01N 33/5011: {for testing antineoplastic activity} - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Embodiments provide methods to produce an assembly that mimics in vitro the human microenvironment of a primary tumor site, a secondary distant organ, and circulation. Methods and apparatuses that provide a platform for the isolation of primary tumor, invasive, and metastatic cells to be used for evaluation of therapeutic efficacy and diagnostics as applied to metastatic cancers are also provided. ## Claims ### Claim 1 A method of identifying an anticancer or anti-hyperproliferative (anti-HPP) therapeutic comprising: a) adding a potential anticancer or anti-HPP therapeutic to a first component of a cell culture assembly comprising (i) the first component comprising a primary site growth medium derived from a vertebrate overlaid on a primary site growth matrix, (ii) a second component comprising a biological matrix mimetic, and (iii) a dynamic fluid component comprising a secondary site growth medium, wherein the dynamic fluid component is in fluid contact with the primary site growth matrix and the biological matrix mimetic, allowing cells to migrate from the primary site growth matrix through the dynamic fluid component to the biological matrix mimetic, wherein the secondary site growth medium has a higher serum concentration than the primary site growth medium, and wherein the primary site growth matrix in the first component comprises a detectable amount of metastatic cancer cells or HPP cells, b) allowing the metastatic cancer cells or HPP cells to migrate from the primary site growth matrix of the first component to the biological matrix mimetic of the second component, c) detecting the amount of metastatic cancer cells or HPP cells present in the biological matrix mimetic of the second component following the migration, and d) identifying the potential anticancer or anti-HPP therapeutic as an anticancer or anti-HPP therapeutic if the addition thereof in the first component reduces the migration as compared to when no such addition is made. ### Claim 2 (depends on 1) The method of claim 1, wherein the cell culture assembly further comprises a system coupled to the assembly for detecting colonization of metastatic cells. ### Claim 3 (depends on 1) The method of claim 1, wherein the cell culture assembly further comprises a digital microscope or a system coupled to the assembly for detecting colonization or growth of metastatic cancer or HPP cells. ### Claim 4 (depends on 1) The method of claim 1, wherein the cell culture assembly further comprises a flow cytometer coupled to the assembly. ### Claim 5 (depends on 1) The method of claim 1, wherein the secondary site growth medium contains at least 8% serum or serum substitute. ### Claim 6 (depends on 1) The method of claim 1, wherein the secondary site growth medium contains at least 15% serum or serum substitute. ### Claim 7 (depends on 5) The method of claim 5, wherein the primary site growth medium contains less than 5% serum or serum substitute. ### Claim 8 (depends on 5) The method of claim 5, wherein the secondary site growth medium has serum concentration that is at least twice of that in the primary site growth medium. ### Claim 9 (depends on 1) The method of claim 1, wherein the first component and the second component are contained within separate tissue culture vessels. ### Claim 10 (depends on 1) The method of claim 1, wherein the first component is at least partially contained within an insert vessel. ### Claim 11 (depends on 1) The method of claim 1, wherein the cell culture assembly further comprises a pump or an agitation apparatus coupled to the dynamic fluid component. ### Claim 12 (depends on 1) The method of claim 1, wherein the primary site growth medium in the first component is derived from a healthy vertebrate. ### Claim 13 (depends on 1) The method of claim 1, wherein the primary site growth medium in the first component is derived from a vertebrate with cancer. ### Claim 14 (depends on 1) The method of claim 1, wherein the dynamic fluid component comprises a fluid obtained from a vertebrate with cancer. ### Claim 15 (depends on 1) The method of claim 1, wherein the dynamic fluid component comprises a fluid obtained from a culture of bone marrow stromal cells. ### Claim 16 (depends on 1) The method of claim 1, wherein the biological matrix mimetic comprises organ-specific matrix. ### Claim 17 (depends on 16) The method of claim 16, wherein the organ-specific matrix simulates the adrenal gland, bone marrow, brain, liver, lung tissue, lymph node, ovary, peritoneum, skin, spleen, connective tissue, bone, vascular structure, or articular joint. ### Claim 18 (depends on 1) The method of claim 1, wherein the biological matrix mimetic comprises collagens 1-14 or fragments thereof, elastin, laminin, fibronectin, hyaluronic acid or related hyaluronans, lecticans, glycosaminoglycans, chondroitins, dermatans, or related extracellular matrix or glycocalyx components or combinations thereof. ### Claim 19 (depends on 1) The method of claim 1, wherein the primary site growth matrix comprises collagen I, collagen II, collagen III, collagen IV, collagen-V, elastin, laminin, fibronectin, hyaluronic acid, lecticans, glycosaminoglycan or combinations thereof. ### Claim 20 A method of identifying the efficacy of an anticancer or anti-hyperproliferative (anti-HPP) therapeutic comprising: a) adding an anticancer or anti-HPP therapeutic to a first component of a cell culture assembly comprising (i) the first component comprising a primary site growth medium derived from a vertebrate overlaid on a primary site growth matrix, (ii) a second component comprising a biological matrix mimetic, and (iii) a dynamic fluid component comprising a secondary site growth medium, wherein the dynamic fluid component is in fluid contact with the primary site growth matrix and the biological matrix mimetic, allowing cells to migrate from the primary site growth matrix through the dynamic fluid component to the biological matrix mimetic, wherein the secondary site growth medium has a higher serum concentration than the primary site growth medium, and wherein the primary site growth matrix in the first component comprises a detectable amount of metastatic cancer cells or HPP cells, b) allowing the metastatic cancer cells or HPP cells to migrate from the primary site growth matrix of the first component to the biological matrix mimetic of the second component, c) detecting the amount of metastatic cancer cells or HPP cells present in the biological matrix mimetic of the second component following the migration, and d) identifying the efficacy of the anticancer or anti-HPP therapeutic based on the therapeutics' impact on the migration as compared to a control.