Poly cystic kidney disease (PKD) is characterized by distribution of a large number of cysts in significantly enlarged kidneys. These cysts cause impairment of kidney function and can eventually cause kidney failure. In humans, PKD can be inherited in autosomal dominant (ADPKD) or autosomal recessive (ARPKD) forms. ADPKD is the most common dominantly inherited kidney disease in humans and the leading genetic cause of eventual dialysis or transplantation. In ADPKD, fluid-filled cysts develop and enlarge in both kidneys, eventually leading to kidney failure. ADPKD is the fourth leading cause of kidney failure and more than fifty percent of people with ADPKD will develop kidney failure by age 50. Once a person has kidney failure, dialysis or a kidney transplant are the only options for survival. Currently, the field lacks a practical solution for identifying agents that can treat or prevent PKD.
Provided herein is a method of producing immortalized, cystogenic, single cyst-derived cells from a polycystic kidney. The method includes the steps of (a) isolating one or more cells from a single cyst in a polycystic kidney; (b) culturing the isolated one or more cells in a culture dish or well under conditions for expanding the one or more cells to create a plurality of single cyst-derived cells; (c) transferring the plurality of single-cyst derived cells to a three dimensional culture system configured for cyst formation; (d) optionally, transferring the plurality of single-cyst derived cells to a three dimensional culture system configured for cyst formation; (e) determining whether the plurality of single-cyst derived cells form cyst-like structures in the three dimensional culture; and (f) immortalizing one or more of the plurality of single-cyst derived cells that form cyst-like structures.
Further provided is a population of immortalized, cystogenic, single cyst-derived cells from a polycystic kidney. Also provided is a three dimensional culture of immortalized, cystogenic, single cyst-derived cells from a polycystic kidney comprising (a) a population of immortalized, cystogenic, single cyst-derived cells and (b) a three-dimensional gel.
Further provided is a method of screening for an agent that prevents or treats polycystic kidney disease. The method includes the steps of (a) contacting a culture of cystogenic, single cyst-derived cells from a polycystic kidney with an agent to be screened; (b) transferring the contacted cells to a three dimensional culture system configured for cyst formation; and (c) determining the level of cyst-like formations in the three dimensional culture system of the contacted cells after treatment with the agent as compared to a control level. A reduced level of cyst-like formations indicates the agent prevents or treats polycystic kidney disease.
The present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.
PKD is a genetic disorder in which renal tubules become structurally abnormal, resulting in the development and growth of multiple cysts within the kidney. These cysts may begin to develop in utero, in infancy, in childhood, or in adulthood. Cysts are non-functioning tubules filled with fluid, which range in size from microscopic to every large, crushing adjacent normal tubules and eventually rendering the normal tubules non-functional as well. PKD is caused by abnormal genes (PKD1, PKD2 or PKD3) that encode abnormal proteins, which affect tubule development. As used throughout, PKD refers to autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD),
Provided herein are compositions and methods for producing immortalized, cystogenic, single cyst-derived cells from a polycystic kidney. Some methods include the steps of (a) isolating one or more cells from a single cyst in a polycystic kidney; (b) culturing the isolated one or more cells in a culture dish or well under conditions for expanding the one or more cells to create a plurality of single cyst-derived cells; (c) transferring the plurality of single-cyst derived cells to a three dimensional culture system configured for cyst formation; (d) determining whether the plurality of single-cyst derived cells form cyst-like structures in the three dimensional culture; and (e) immortalizing one or more of the plurality of single-cyst derived cells that form cyst-like structures.
In the methods provided herein, the one or more cells are isolated from a single cyst in a polycystic kidney. In some methods, the polycystic kidney is from a human subject. In some methods, one or more pluralities of single-cyst derived cells can be produced from one or more cells isolated from the same single cyst. In other methods, one or more pluralities of single-cyst derived cells can be produced from one or more cells from different single cysts in a polycystic kidney. Methods of isolating or dissecting single cysts from a polycystic kidney are known in the art. See, for example, Carone et al., “Cyst-derived cells do not exhibit accelerated growth or features of transformed cells in vitro,” Kidney International 35: 1351-1357 (1989). Methods for isolating single cysts are also provided in the Examples.
After isolation, the single cyst or tissue from a single cyst can be further processed. For example, the single cyst or tissue from a single cyst can be further processed using mechanical disruption or enzymatic digestion techniques. (See, for example, Carone et al.). The enzymatic digestion protocol described in the Examples can also be used to isolate one or more cells from the single cyst. The isolated one or more cells are then placed in a culture dish or well and expanded to create a plurality of single cyst-derived cells, i.e., a primary cell culture. The culture dish can be a 35 mm tissue culture plate, a 60 mm tissue culture plate, a 100 mm tissue culture plate, a 150 mm tissue culture plate tissue culture plate, or a larger tissue culture plate. The cells can also be placed in a 6-well, 12-well, 24-well, 48-well, 96-well, 192-well, 384-well or 768-well tissue culture plate. The cells can also be expanded in a flask, for example, in a T-25, a T-75 or a T-175 flask. In any of the methods provided herein, the cells can be expanded until the dish, well, plate, flask etc., is at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% confluent.
Optionally, the isolated one or more cells are placed on a permeable filter support-based culture and expanded to create the primary cell culture.
As used throughout, the phrase primary, in the context of a primary cell, refers to a cell that has not been transformed or immortalized. Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some cases, the primary cells are adapted to in vitro culture conditions. In some cases, the primary cells are stimulated or activated.
In some methods, the one or more cells isolated from a single cyst of a polycystic kidney can include one or more fibroblasts. Therefore, during expansion, the plurality of single cyst-derived cells can include fibroblasts. In some examples, the plurality of single cyst-derived cells comprises less than about 10%, less than about 5%, or less than about 1% fibroblasts. Optionally, the one or more cells are cultured in media that maintains the epithelial phenotype of the single cyst-derived cells. Optionally, the one or more cells are cultured in media that maintains the epithelial phenotype of the single cyst-derived cells and increases the proliferation rate of the cells. In some examples, the proliferation rate is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or 400% as compared to a plurality of cyst-derived cells not contacted with the media.
Once the one or more cells single-cyst derived cells are expanded to create a plurality of single cyst-derived cells, the plurality is transferred to a three dimensional (3D) culture system configured for cyst formation. Optionally, the 3D culture system comprises media that maintains the epithelial phenotype of the single cyst-derived cells and/or increases the proliferation rate of the one or more cells. Since 3D cell culture systems more accurately represent the microenvironment where cells reside in tissues, the behavior of 3D cultured single cyst-derived cells is more reflective of in vivo cellular responses, for example, cyst formation by kidney cells in kidney parenchymal tissue.
While a traditional two-dimensional culture usually grows cells into a monolayer on glass or, more commonly, in tissue culture polystyrene plastic flasks, 3D cell cultures grow cells into 3D aggregates/spheroids using a scaffold/matrix or in a scaffold-free manner. Scaffold/matrix-based 3D cultures can be generated by seeding cells on an acellular 3D matrix or by dispersing cells in a liquid matrix followed by solidification or polymerization. Commonly used scaffold/matrix materials include biologically derived scaffold systems and synthetic-based materials. Commercially available products such as BD Matrigel™ basement membrane matrix (BD Sciences, Franklin Lakes, N.J.), Cultrex® basement membrane extract (Trevigen, Gathersburg, Md.), and hyaluronic acid are commonly used biologically derived matrices. Polyethylene glycol (PEG), polyvinyl alcohol (PVA), polylactide-co-glycolide (PLG), and polycaprolactone (PLA) are common materials used to form synthetic scaffolds. Scaffold-free 3D cell spheroids can be generated in suspensions by the forced floating method, the hanging drop method, or agitation-based approaches. See, for example, Breslin et at, “Three-dimensional cell culture: the missing link in drug discovery,” Drug Discov. Today 18: 240-249 (2013); Gurski et al., “Three-dimensional matrices for anti-cancer drug testing and development,” Oncol. Issues 25: 20-25 (2010); and Edmonson et al., “Three-dimensional cell culture systems and their applications in drug discovery and Cell-Based Biosensors,” Assay Drug Dev. Technol. 12(4): 207-218 (2014). Using 3D cell culture methods, cells grow naturally in a 3D environment, allowing cells to interact with each other, the extracellular matrix (ECM), and their microenvironment. In turn, these interactions in such 3D spatial arrangement affect a range of cellular functions; including cell proliferation, differentiation, morphology; gene and protein expression, and cellular responses to external stimuli.
Optionally, the transferred plurality of single-cyst derived cells have been passaged fewer than five times prior to transfer. Optionally, the transferred plurality of single-cyst derived cells have been passaged fewer than three times prior to transfer. Optionally, the lifetime of any of the primary cultures described herein can be extended by one, two, three, four or more passages, by growing the primary single-cyst derived kidney cells on permeable filter supports prior to seeding the cells into a 3D tissue culture system. In some cases, the cells can be grown in tissue culture plates, wherein each well of the tissue plate contains a clear polyester filter support, which allows visualization of the cells during expansion. In other cases, the cells can be grown on a dish permeable filter support, for example, a support that is made from polycarbonate. Optionally, primary cultures that struggle to form cysts are stimulated with forskolin and/or arginine vasopressin (AVP), which mobilize cyclic AMP (cAMP), or with growth factors like epidermal growth factor (EGF) to trigger cystogenesis in primary cultures that do not form cysts in media alone. In some methods, the 3D tissue culture system includes forskolin. AVP, and/or EGF to enhance cyst formation.
In some methods, the plurality of single-cyst derived cells are transferred to or embedded in a 3-D culture system for an amount of time sufficient to allow cyst formation. For example, the cells can be in the 3-D culture system for one, two, three, four, five, six, seven, eight, nine, ten or more days before and while cysts appear. If cyst formation occurs in the 3D culture system, with or without stimuli, the plurality of cells from a sample of the primary culture, prior to 3D culturing is a cyst-forming plurality that can be immortalized. These immortalized cell lines can be used in assays to identify agents that treat or prevent kidney cyst formation.
In the methods provided herein, one or more cells of the plurality of single-cyst derived cells that form cyst-like structures can be immortalized using methods known in the art. For example, and not to be limiting, the cells can be transfected with a viral oncogene, for example, SV40 and/or a human telomerase (hTERT). See, for example, Sarrab et al., “Establishment of conditionally immortalized human glomerular mesangial cells in culture, with unique migratory properties,”Am. J. Physiol. Renal Physiol. 301(5): F1131-F1138 (2011); Lechner et al., “Human epithelial cells immortalized by SV40 retain differentiation capabilities in an in vitro raft system and maintain viral DNA extrachromosomally,” Virology 185(2): 563-71 (1992); and Lee et al., “Use of exogenous hTERT to immortalize primary human cells,” Cytotechnology 45(1-2): 33-38 (2004)). Other methods involve CDK4 or CDNK2 genetic constructs alone or in combination with the hTERT method of immortalization.
In some methods, one or more cells isolated from a single cyst are genotyped and/or whole exome sequenced. Genotyping allows identification of a primary germline PKD1 mutation(s) that is present in all cells from a single cyst as well as any somatic PKD1 mutations present in one or more cells of the single cyst, Methods for genotyping kidney cells are known in the art and include, but are not limited to, restriction fragment length polymorphism identification (RFLPI) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism detection (AFLPD), polymerase chain reaction (PCR), DNA sequencing (for example, whole genome or whole exome sequencing), allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads.
Examples of germline mutations in PKD1 include, but are not limited to, a P2736R mutation, a L2763V mutation, a M2764T mutation, a frameshift/truncation at E685, a P872X ns/truncation, a R2767 frameshift/truncation, and a G3690E missense mutation. Examples of somatic PKD1 mutations include, but are not limited to, a P3448 frameshift mutation, a A62 frameshift mutation, a L237 frameshift mutation, a L715 frameshift mutation, a Q160 frameshift mutation, a K718 frameshift mutation, a Q542 frameshift mutation, a Y684 frameshift mutation, a N116 frameshift mutation and a G617 frameshift mutation. By genotyping the cells isolated from a single cyst, the genotyped cells can be used to identify agents that treat or prevent cyst formation associated with the specific mutation(s) in the PKD1 gene and/or the PKD2 of the cell.
Also provided is a primary culture of cystogenic, single cyst-derived cells from a polycystic kidney. Optionally, the primary culture of cystogenic, single cyst-derived cells can be made using any of the methods described herein. Optionally, the primary culture of cystogenic, single cyst-derived cells is produced by (a) isolating one or more cells from a single cyst in a polycystic kidney; (b) culturing the isolated one or more cells in a culture dish or well under conditions for expanding the one or more cells to create a plurality of single cyst-derived cells; (c) transferring a first subset of the plurality of single-cyst derived cells to a three dimensional culture system configured for cyst formation; (d) determining the first subset of the plurality of single-cyst derived cells form cyst-like structures in the three dimensional culture; and (e) isolating a second subset of the plurality of single cyst-derived cells to create a primary culture of cystogenic, single cyst-derived cells from a polycystic kidney. Optionally, any primary culture described herein or made by a method described herein can be immortalized.
Further provided are populations of cells passaged from a primary culture described herein. Optionally, the population of cells is a population that has been passaged one, two, three, four, five or more times. Optionally, the population of cells is a population that has been passaged fewer than five, four, three or two times.
Also provided is a population of immortalized, cystogenic, single cyst-derived cells from a polycystic kidney. Optionally, the population is a population derived using any of the methods for producing an immortalized cystogenic, single cyst-derived cells as described herein. Also provided is a 3D culture of immortalized, cystogenic, single cyst-derived cells from a polycystic kidney comprising (a) a population of immortalized, cystogenic, single cyst-derived cells and (b) a 3-D gel. In some examples, the population of immortalized, cystogenic, single cyst-derived cells in the 3D culture is a population of immortalized, cystogenic single cyst-derived cells produced by any of the methods provided herein.
Specific immortalized, cystogenic, single cyst-derived cells from a polycystic kidney are useful for transepithelial electrical assays such as transepithelial electrical resistance and voltage assays under open-circuit conditions, fluid transport assays, and Ussing chamber-driven electrophysiology. The cells are growth on permeable filter supports for these bioassays.
Any of the primary or immortalized cell cultures described herein can be used to identify one or more agents that decrease or reduce cyst-like formation. A decrease in cyst-like formation can be a decrease in the number of cysts, a decrease in growth of an existing cyst, a decrease in the growth rate of an existing cyst, or a delay in the appearance of one or more cysts. Provided herein is a method of screening for an agent that prevents or treats polycystic kidney disease comprising (a) contacting a culture of cystogenic, single cyst-derived cells from a polycystic kidney with an agent to be screened; (b) transferring the contacted cells to a three dimensional culture system configured for cyst formation; and (c) determining the level of cyst-like formations in the three dimensional culture system of the contacted cells after treatment with the agent as compared to a control level. A reduced level of cyst-like formations indicates the agent prevents or treats polycystic kidney disease.
Further provided is a method of screening for an agent that prevents or treats polycystic kidney disease comprising (a) contacting a 3D culture system of cystogenic, single cyst-derived cells from a polycystic kidney with an agent to be screened and (h) determining the level of cyst-like formations in the three dimensional culture system of the contacted cells after treatment with the agent as compared to a control level. A reduced level of cystogenic capacity indicates the agent prevents or treats polycystic kidney disease.
Optionally, the 3D culture system of cystogenic, single cyst-derived cells from a polycystic kidney is contacted with the agent after two or more cysts have formed in the 3D culture system, for example, after one, two, three, four, five, six, seven, eight, nine, ten or more days of cell growth in the 3D culture system. Once sizable cysts have formed, the 3-D system is contacted with the agent and the level of cyst-like formations in the 3D culture system is determined. A reduced level of cystogenic capacity, for example, fewer cysts and/or smaller cysts, as compared to a control, indicates the agent treats polycystic kidney disease.
The level of cystogenic capacity can be determined 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days or later after contacting the 3D culture system with the agent. The reduction in cystogenic capacity does not have to be complete as this can range from a decrease in cytogenic capacity to complete ablation of one or more existing cysts. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels.
Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., polycystic kidney disease, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom, for example, reduction in the size of one or more cysts in the kidney of a subject. Treating or treatment includes modulating the disease or disorder. Treating or treatment includes delaying or preventing progression of polycystic kidney disease. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
Optionally, the 3D culture system of cystogenic, single cyst-derived cells from a polycystic kidney is contacted with the agent before or when cysts have just formed in the 3D culture system, for example, before one, two, three, four, five, six, seven, eight, nine or ten days of cell growth in the 3D culture system. Optionally, the culture system is contacted with the agent when two or more cysts have first formed. Optionally, when performing the assay in multiple yells or dishes, the culture system is contacted with the agent when an average of three cysts per culture system first form. The level of cystogenic capacity can be determined 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days or later, after contacting the 3D culture system with the agent. A reduced level of cystogenic capacity, for example, fewer cysts, or smaller cysts, as compared to a control, indicates the agent prevents the development of polycystic kidney disease. In some examples, the control is the cystogenic level of untreated cells derived from the same cyst
Optionally, any of the screening methods provided herein can further comprise contacting normal human kidney cells with the agent to determine the toxicity of the agent. For example, when transferred or embedded in a 3D culture system, normal human kidney cells generate tubule or duct-like networks. Sometimes, the normal human kidney cells form small cysts and spheroids. High content imaging is performed to visualize the cysts, spheroids, tubules and other structures daily and over time. In methods comprising screening for toxicity, one or more agents can be contacted with a 3D culture system comprising normal human kidney cells and the level of tubulogenesis can be assessed, prior to screening the agent for cystogenic activity. Optionally, if the agent is toxic, the agent is not screened for cystogenic activity. A 50% reduction in tubulogenesis to elimination of tubule structures is considered cytotoxic in this assay. These assays thus identify cytotoxic agents and chemicals or small molecules that are toxic to renal cells and tissues.
Optionally, any of the screening methods provided herein can further comprise determining the level of inflammatory factors secreted by the cells after being contacted with the agent. A decrease in the production or secretion of one or more inflammatory factors indicates that the agent inhibits inflammation associated with polycystic kidney disease. Monocyte chemoattractant protein 1 (MCP-1), interleukins 8 and 6 (IL-8 and IL-6), and tumor necrosis factor alpha (TNFalpha) are commonly hypersecreted by cystogenic, single cyst-derived cells from a polycystic kidney.
In any of the screening methods provided herein, the agent can be, but is not limited to, a chemical, a small or large molecule (organic or inorganic), a drug, a protein, a peptide, a cDNA, an antibody, a morpholine, a triple helix molecule, an siRNA, a shRNA, an miRNA, an antisense RNA or a ribozyme to name a few. Optionally, in any of the screening methods provided herein, the culture of cystogenic, single cyst-derived cells from a polycystic kidney comprises a primary culture of cells isolated from a single cyst or immortalized cells. Optionally, the culture comprises immortalized, cystogenic, single cyst-derived cells from a polycystic kidney Optionally, in any of the screening methods provided herein, the method can further comprise correlating the agent that prevents or treats polycystic kidney disease with a specific genetic profile of the cystogenic, single cyst-derived cells. Optionally, in any of the screening methods provided herein, the control level is the cystogenic level of untreated cells derived from the same cyst.
Disclosed are materials, compositions, and ingredients that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods.
500 ml bottles of Ca2+/Mg2+−free phosphate buffered saline (PBS) (or Hanks' Balanced Salt Solution (HBSS)), with 5× antibiotics ((25 mls of 100× Pen/Strep stock solution (Corning Fisher Scientific (Hampton, N.H.), 10 mls of Fungizone (Amphotericin B) stock solution (Corning Fisher Scientific), and 1 ml of Gentamicin stock solution (Corning Fisher Scientific) were prepared.
At least two liters of Ca2+/Mg2+− free DMEM/F-12 Ham's media (14.8 grams of powder media were mixed with 1 liter of distilled water and filter sterilized with a 1 liter filter bottle system) (Sigma-Aldrich (St. Louis, Mo.)) with 5× antibiotics (as above), with 5 mls of added L-Glutamine 100× stock solution were prepared.
500 ml bottles of Advanced MEM (Gibco-BRL (Hampton, N.H.), Invitrogen (Carlsbad, Calif.)), with 5% FBS, 5× antibiotics (as above) and 5 mls of added L-Glutamine 100×, stock solution were prepared. This media was used for washing the tissues after each digestion.
At least one liter of Ca2+/Mg2+− free DMEM/F-12 Ham's media with 5× antibiotics (as above), L-Glutamine (as above) and 0.5 nag/ml Collagenase P (Roche, Basel, Switzerland) was prepared to create Digestion Media for 4° C. and 37° C. digestions.
Several liters of DBM RenalCyte media supplemented with 5× antibiotics were prepared.
The human kidney tissue specimen was removed from triple-bagged containers on ice. Excess liquid was aspirated. Ca2+/Mg2+− free PBS was poured over the tissue to wash/moisten the tissue. Any excess adipose tissue was dissected away from the tissue. This was minimal to quite substantial and was donor dependent.
End-stage human ADPKD kidney tissue fragments or whole kidneys were quite large and fully compromised with fluid-filled cysts. The large cysts that can be individually dissected were readily apparent. The initial dissection emphasized isolating as many single cysts as possible, with each cyst going into their own separate culture vessel, immediately after dissection. Each cyst was kept separate, in primary culture, from that point on. The steps below describe the dissection of a single isolated cyst.
Large cysts were individually dissected from the tissue. Often, course scissors were needed to cut through tough connective tissue surrounding the largest cysts. Cysts often held clear fluid, blood or cloudy fluid. Collection of clear fluid-filled cysts were prioritized but blood colored fluid-filled cysts were also collected. Cloudy fluid-filled cysts were generally avoided.
Each single cyst was placed into its own Petri dish and washed again with Ca2+/Mg2+−−free PBS with 5× antibiotics. Extraneous tissues were then dissected from the surface of each cyst, and any fluid inside the cysts was dispelled. Upon removal of fluid, the cyst collapsed. The cyst was washed again with Ca2+/Mg2+−+-free media including 5× antibiotics and partially Minced. Then, the cyst wall tissue fragment was placed into cold Digestion Media, typically, in a 50 ml conical tube, and the minced tissue from each cyst was placed into 20 ml of Digestion Media. The cyst tissue was incubated in Digestion Media, in the 50 ml conical tube, overnight at 4° C. This procedure was repeated for as many individual single cysts as possible with Ca2+/Mg2+−free PBS poured over the tissue many times during dissection. Once all desired single cysts were individually dissected and placed into separate Petri dishes and conical tubes, multicystic, microcystic tissue sections were dissected out of the donor kidney tissue.
Sections of multicystic/microcystic kidney tissue were dissected out. Typically, these were clusters of very small cysts within the tissue. A section of such tissue was placed into a Petri dish. The section was washed with Ca2+/Mg2+−-free PBS and any excess adipose tissue or connective tissue was dissected away. The tissue was washed again with Ca2+/Mg2+−-free media and minced with scissors. The minced tissue was then placed into a 50 ml conical tube filled with 20 mls of Digestion Media and incubated overnight at 4° C.
Normal and diabetic human kidney specimens were closer to normal in size, although diabetic human kidneys can display hypertrophy and become larger. After washing, as described above, the kidney was cut longwise and in half down the long axis of the kidney, to obtain two halves of the kidney with the inner regions of the kidney exposed. The inner medulla, the white matter near the point where the collecting ducts coalesce, was dissected away from the rest of the kidney tissue and processed as inner medulla primary cultures. The outer medulla, (the pink or red portions where the pyramidal calyxes of the medulla reside) was dissected separately and processed into its own primary culture. The majority of the kidney tissue (that which remained after dissecting away medullary regions) was the cortex of the kidney. The cortex was dissected in sections or slices and processed into primary cultures. In some procedures, there were about two to four Petri dishes of inner medulla tissue, three to six Petri dishes of outer medulla tissue, and as many as 16 Petri dishes of cortex to process. Each section of tissue was placed into a Petri dish and washed with Ca2+/Mg2+−free PBS. Any excess adipose tissue or connective tissue was dissected away prior to washing the tissue again with Ca2+/Mg2+−free media. Scissors were used to mince the tissue. The minced tissue was placed into a 50 ml conical tube filled with 20 mls of Digestion Media and incubated overnight at 4° C.
Primary Cell Collections from Tissue Digestions
For all types of tissue digested from diseased or normal human kidneys, the following step-wise process was used for collecting cell pellets from the digestions over time.
Tubes were removed from the refrigerator and 5 mls of straight fetal bovine serum (FBS) were added to each tube to stop collagenase P activity. Gentle agitation was used to mix in the FBS. Then, each 50 ml conical tube with digesting tissue was inverted 20-25 times to agitate before allowing the tissue to settle to the bottom of the tube. The supernatant was pipetted off and 20 mls of Advanced MEM (plus 5% FBS plus 5× antibiotics) were added to each conical tube with tissue pieces. Each tube was inverted again about 20-25 times to agitate and supernatant was again removed and collected with the first supernatant. The supernatants were centrifuged for 5 minutes at 2400 RPM. Large cell pellets, with a red coloring due to the presence of RBCs, appeared at the bottom of the tube. Most of the supernatant was removed, leaving behind a bit of liquid to break apart the cell pellet with vortexing. One ml of DBM RenalCyte media with 5× antibiotics was added to resuspend the cells with a bit of fresh media to rest the cells before seeding into T175 flasks. The entire cell pellet was seeded into a T175 flask. This was done for each cell pellet for each primary cell culture being generated.
A second digestion was performed at 37° C., in a cell culture incubator, for one hour. The steps of the first digestion were repeated. A third digestion was performed at 37 CC in a cell culture incubator for another hour. Twenty mls of Ca2+/Mg2+−free DMEM/F12 (Digestion Media) with 0.5 mg/ml Collagenase P kept at room temperature were added to the tubes still containing the tissue to be digested. Then, the tubes were placed in the cell culture incubator for a final hour and the steps of the first digestion were repeated.
At this point, there were 3 T175 flasks for each primary culture established. In some cases the digested tissue was pipetted up and down to break it apart and smaller pieces that could be pipetted into a 4th T175 flask were seeded to establish a Cyst 1 Explant culture.
The T175 flasks were placed in incubators and left undisturbed for four days to allow all viable cells to attach well to the substrate. Small tissue fragments also attached and cells grew out. After the 4-day cell attachment phase, the flasks are washed with Ca2+/Mg2+−containing HBSS with 5× antibiotics to clean the primary cultures of any and all unattached debris. As many as three washes were required to clean the primary cultures. At this point, attached and viable cells were visible. Images were taken and percent confluence was assessed.
After the washes, fresh DBM RenalCyte media was added (20-24 mls) to re-feed the cultures. Cells reached confluence as fast as 8-9 days after seeding, but in some cases took 2-3 weeks. Once a T175 flask was confluent, the flasks were washed once in Ca2+/Mg2+−-free PBS with 5× antibiotics and then incubated in 18 mls of the same for at least 1 hour in a 37° C. incubator. After the Ca2+/Mg2+−free PBS incubation step, the flasks were hit multiple times to loosen the attached cells. Loosened cells were collected in a 50 nil conical tube and 10 rats of DBM RenalCyte media were added to keep the cells fed. Then, 12 mls of trypsin-EDTA solution were added. The flasks were then incubated until all of the cells attached. After the trypsin-EDTA incubation step, the flasks were hit multiple times and loosened cells were collected in the same 50 ml conical tube with the PBS-loosened cells and media. Afterwards, the cells were pelleted by centrifugation for 5 minutes at 2400 RPM. The supernatant was poured off, but a little liquid was left to break apart the cell pellet with vortexing. One ml of DBM RenalCyte media was added. A 5 μl aliquot of the cell suspension was mixed with 5 μl of Trypan Blue and counted in the Invitrogen Countess II machine. At this point, an aliquot of the cells was seeded into a new flask for passage 1 or the cells were used in an assay. A 10 μl aliquot of the cells was pipetted into a separate small tube and mixed with 300 μl of cold DBM 3D Biogel solution to form a 3D Biogel. The cells were then tested for cystogenic or tubulogenic phenotype.
Upon cell counting, there were often at least 10 million cells derived from a very-confluent T175 flask, although this number varied. With the cell pellet already resuspended in 1 ml of DBM RenalCyte media, cold DBM Cryopreservation Media was added, using 1.5 mls for every cryovial. Generally there were greater than one million cells per cryovial, but this number can vary. DBM Cryopreservation Media, Lifeline Cell Technology's FrostaLife (Oceanside, Calif.) or Sigma-Aldrich's CryoSOfree media can be used.
The lifetime of the primary cultures grown on standard tissue culture plasticware is about four passages. However, by growing the primary huADPKD cystic kidney cells on permeable filter supports, the lifetime of the same primary cultures is extended to at least eight passages. The practice of growing and maintaining primary huADPKD cystic cells and primary nominal human kidney cells on permeable filter supports also helps maintain the ability of the cells to form cysts or tubules, respectively, in a 3D tissue culture system, for example a 3D Biogel. In some cases, the cells were grown on Corning Costar 3450-6 plates wherein each well contains a large 24 mm clear polyester filter support, which allows visualization of the cells during expansion. The cells were optionally grown on a Corning 2419 10 cm dish permeable filter support made from polycarbonate.
Two 10-lb diseased human ADPKD) (huADPKD) kidneys from a donor were received and processed into 50 single cyst-derived primary cultures and 8 multicystic microcystic tissue-derived primary cultures. In total, approximately 1,500 cryovials of 1 million cells per cryovials were expanded and cryopreserved as passage 0 and passage 1 cells. With standard re-derivation on permeable filter supports, these primary cultures can be well maintained as early passage cells.
The second digestion and second wash huADPKD cell pellets yielded better primary cell yield in seeding large T175 flasks. The first digestion and first wash yielded cells as well; however, the cultures were slower to reach confluence and had higher fibroblast content.
There were 4 groups or waves of cells that were fast to slow in terms of being cryopreserved and seeded into 3D biogels to assess cystogenic phenotype. In general, the first two ‘waves’ of primary cultures were cystogenic and displayed the proper mixture of cell types. The slower two waves' had higher fibroblast content and may need to be subjected to differential trypsinization to lower the fibroblast number; but, many of these cultures still made cysts. Those cultures that are too fibroblastic can be used to assess lead therapeutic assets for aggressive hyperproliferative fibroblast anti-proliferation assays. Overall, after assessing cystogenecity, there were many primary cultures (the majority) that were cystogenic. Those deemed ‘OK with higher than desirable fibroblast content’ were subjected to differential trypsinzation and improved to cystogenesis assays. Those with an unacceptably high fibroblast percentage can be used for anti-proliferation assays. Some of the cultures were used to assay for secreted chemokine inhibition and for anti-proliferative effects against aggressive hyperproliferative fibroblasts after administration of a test compound.
High-titer aliquots (109 pfu) of Lenti-SV40T, Lenti-hTERT-Neo and Lenti-CDK4 from Applied Biological Materials (ABM) (Richmond, BC, Canada) and Lenti-CDNK2 (Sigma-Aldrich) were diluted into transfection media to 107 pfu for cell challenge. In some cases, Lenti-hTERT and Lenti-CDK4 or Lenti-CDNK2 were combined as a newer immortalization method. huADPKD primary cultures were selected for immortalization based on a compelling genotype, an acceptable cell proliferation rate, and an ability to form cysts in 3D Matrigel culture. Mixed immortalized cultures were established by both methods from primary cultures where the lifetime of the cells were doubled from 5 passages (primary) to 10 passages (mixed immortal). The SV40T method was successful in generating immortal cell cultures that formed electrically tight polarized cell monolayers for transepithelial resistance (TEER) measurements and likely Ussing chamber-based electrophysiological recording of ion transport. These immortal human ADPKD cell monolayers may also be useful in fluid transport assays. Transepithelial/transendothelial electrical resistance (TEER) data for a new immortalized human ADPKD cell line are shown in Table 1 below. The increase in resistance with amiloride treatment shows that the epithelial sodium channel (ENaC) is expressed in the cell monolayers.
With the Human Genetics Cores in the Mayo Clinic PKD Center and at the University of Arizona, flash frozen cell pellets of our primary human ADPKD cell cultures (mainly, the single cyst-derived cultures) were sent to the Cores, who isolated the genomic DNA and either used the Sanger method of DNA sequencing or next generation sequencing of the PKD11, PKD2, and PKHD1 genes or, in more rare cases, whole genome sequencing (WGS) if it was unclear as to whether the kidney was a bona fide polycystic kidney. Table 2 shows the results of genotyping studies.
$Donors 8 and 11 both had regions or ‘pockets’ within the diseased kidney that were non-cystic. This is difficult to identify/dissect from later stage or end stage ADPKD tissue specimens.
As shown in Table 2, twelve donors have been processed. In Table 2, SC=single cyst-derived primary culture; MCT=microcystic multicystic tissue-derived primary culture; and NHK=primacy cultures derived from non-cystic tissue identified within an ADPKD tissue specimen.
Single cyst-derived cultures were genotyped for germline mutations as well as any secondary somatic mutations. The results for seven donors show the following: no somatic mutations were found in 3 donors. 1-2 instances of somatic mutations were found in 2 donors, and one single PKD2 mutant-bearing donor had frequent examples of secondary somatic hits which were different from individual cyst culture to cyst culture. The mutations are donor dependent. In some instances, ADPKD patients may have efficient enough gene editing to prevent somatic mutations while others may not.
Primary cells capable of 3D cystogenesis were seeded into a 3D biogel that is liquified before the gel sets. The gel set undisturbed within a humidified 5% CO2 incubator for 18 hours. An equal volume of DBM RenalCyte media was then overlaid in each well of a 48-well plate. “Add” feeds were performed until the well was full, and then half of the volume of media was removed and new media added at the same volume thereafter. Seven to ten days were required for cysts to appear (based on visual inspection with light microscopy). In the cyst prevention assay, cysts were challenged as soon as they started to appear (small in dimension and few in number per well) with a test compound. The timing of addition of the test compound, frequency of addition of the test compound and concentration of the test compound varied. The effects on the cysts were followed by light-based microscopic imaging over time. In addition to visual inspection by light-based microscopic imaging occurring throughout the study, the number of cysts were counted per well at the end of the study and light-based endpoints were examined, such as 3D CellTiter-GLO® from Promega (Madison, Wis.) (to quantify viable cells), secretion (to quantify cell membrane integrity), and/or Caspase-GLO® (Promega) (to measure induction of apoptosis) in multiplex fashion. A modified version of this assay images after seeding the biogel each day for complete prevention of cyst formation versus counterpart controls over a 14 to 21-day period. Multiple replicates were performed, and cells from multiple donors were tested.
As shown in
DMSO vehicle controls at concentrations of 1-30 μM were performed. Results are shown in
In another exemplary cyst prevention assay, 3D CellTiter-GLO® was used to quantify viable ADPKD cells in a biogel, after thirteen days of exposure to concentration-response curves of selected compounds in a compound screening assay. The results are shown in
Primary cells capable of 3D cystogenesis were seeded as described above for the cyst prevention cytogenesis bioassay. In the cyst attack assay, cysts were allowed to develop to 12-24 in number and allowed to expand to a significant size. Specific cysts or cyst clusters were marked in each well. A test compound was then added to attack the already formed cysts to determine effects on the cysts. Cysts were followed by light-based microscopic imaging over time. The timing of addition of the compound, frequency of addition of the compound and concentration of the compound varied. In addition to visual inspection by light-based microscopic imaging occurring throughout the study and at study end, and the number of cysts counted per well at study end, light-based endpoints were examined such as 3D CellTiterGLO (to quantify viable cells), LDH secretion (to quantify cell membrane integrity), and CaspaseGLO (to measure induction of apoptosis) in multiplex fashion. Multiple replicates were performed, and cells from multiple donors were tested.
A prominent cyst was imaged before treatment and daily for 6 days after treatment began. This larger cyst increased by 2.5 to 3.0 fold in the presence of the industry standard drug, tolvaptan, a vasopressin receptor antagonist. As shown in
As shown in
A BioTek Cytation 5 (Biotek instruments, Winooski, Vt.) high content imaging and light-based high-throughput screening imaging system was used to image the huADPKD cells growing in 3D Biogels in 96-well and 384-well microliter plates. The Cytation 5 is coupled to a BioTek BioSpa 8 (a humidified CO2 incubator), such that trays can be extracted one at a time for analysis in the reader and then returned to the BioSpa. Cytogenesis was observed from key primary and immortalized huADPKD cell cultures in 96-well full area well plates, 96-well half area well plates and in 384-well plates.
Whole well automated high-content imaging was performed of ADPKD primary cells seeded in 384-well plates using the BioTek Cytation 5 system. Multiple well were captured per treatment. Uniform images were fed into deep learning algorithms for analysis. Stimuli like cAMP agonists and growth factors were not required, as the DBM RenalCyte media was sufficient to drive cystogenesis, as shown in
ADPKD cells were cultured for 14 days in the absence or presence of Arginine Vasopressin (AVP), As shown in
High-content imaging, cell viability analysis, cyst counting, and cyst size analysis were performed on cultures in the presence of an industry standard control drug, ricolinostat (0.3-100 μM). Results are shown in
Compound 1 was tested in both prevention and attack assays. Results are shown in
Compounds 1, 2, and 5 were also compared to Vertex CFTR corrector drugs (10 μM) and to Tolvaptan (10 μM) on primary human ADPKD cultures with multiple enlarged cysts per 3D Biogel in a 3D CellTiterGLO Assay of cell viability. Cultures were incubated for 2 weeks before 1-week treatment with the drugs or DMSO control and media only controls. With Compounds 1 and 2, 60-65% inhibition was observed, whereas Compound 6 (an analog of Compound 1), the Vertex CFTR corrector drugs, and Tolvaptan only showed about 20% inhibition at a 10-fold higher concentration.
Compounds 1, 2, and 6 were compared to Vertex CFTR corrector drugs in a 3D CellTiterGLO assay of cell viability using a CRC design in which ADPKD cell cultures had a few small cysts per 3D Biogel starting to form (i.e., incubated 10 days before 1 week of treatment with drugs or media or DMSO only). Results are shown in
Similarly,
An initial screen of small molecule collections on normal human kidney (NHK) cells grown in 3D Biogels is performed to insure that these molecules are not toxic to NHK cells and that they do not disrupt tubulogenesis in 3D Biogels. Small molecules that are not harmful are assessed in the primary human ADPKD cystogenesis assays described above, to determine if they attenuate cystogenesis over time. Supernatants are collected at study end to determine if production/secretion of select inflammatory mediators is attenuated (so called potential inhibitors of the ADPKD inflammasome). Validated inhibitors of ADPKD cystogenesis and the inflammasome are tested in cells derived from at least 2 additional ADPKD donors.
This application claims the benefit of U.S. Provisional Application No. 62/854,756 filed on May 30, 2019, which is hereby incorporated by reference in its entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/034992 | 5/28/2020 | WO | 00 |
Number | Date | Country | |
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62854756 | May 2019 | US |