Methods for isolating cells from placental tissue

Abstract

Disclosed herein are methods for isolating a population of cells from tissue. Among other things, the disclosure provides a method for isolating cytotrophoblast cells from placental tissue. The method involves separating the population of cells using laboratory equipment coated with a lubricant, such as petrolatum. The methods described herein provide for consistent in vitro isolation of purified cells.

Description

Technical Field

[0001] (Description of Federally Sponsored Research or Development) The present invention was made with government support under grant number S21MD000104 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. As used herein, "government" means the United States of America government.

[0002] The present disclosure generally relates to methods of isolating a population of cells from tissue, and more specifically to methods of isolating trophoblasts from placental tissue.

Background Art

[0003] The placenta is a fetal organ that is involved in nutrient and gas exchange between the mother and fetus throughout pregnancy. Abnormal placental formation can cause various pregnancy complications (e.g., miscarriage, preeclampsia, and intrauterine growth restriction), which increases the risk of developing serious disorders (e.g., cardiovascular disease and type 2 diabetes) later in life. Therefore, understanding the molecular mechanisms that govern human placental formation and the trophoblast cell lineage is important.

[0004] Numerous conventional cell culture techniques exist that use various types of placental-derived cells to understand placental biology. However, these techniques lack the organ-specific ultrastructure and physiological function of the placenta. To deepen our understanding of placental biology in human development, human placenta modeling has progressed to the establishment of a placenta-on-a-chip model that enables real-time monitoring based on in vitro compartmentalization of primary human placental cells (Mittal R et al., J Cell Physiol. 2019;234(6):8352~8380; Lee JS et al., J Matern F et al., Neonatal Med. 2016;29(7):1046~54; Yin F et al., Toxicol in vitro. 2019;54:105~113; Blundell C et al., Adv Healthc Mater. 2018;7(2)). These microdevices are created using microfluidic elements, microfabrication techniques, and microsystems containing two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane (Lee JS et al., J Matern Fetal Neonatal Med. 2016;29(7):1046~54). Essential cellular components of these typical placental microsystems are placental trophoblasts.

[0005] The widespread availability of commercially available trophoblast cell lines derived from human choriocarcinoma, such as BeWo, JEG-3, and JAR, has been useful. However, some of these transformed cell lines have been cultured for decades and do not mimic primary trophoblasts in vivo. (Pattillo RA et al., Cancer Res. 28:1231~1236, 1968; Pattillo RA et al., Ann. NYAcad. Sci. 172:288~298, 1971; Kohler PO et al., J. Clin. Endocrinol. 32:683~687, 1971; Pattillo RA et al., In Vitro 6:398~399, 1971). The process of isolating primary trophoblasts from placental tissue is time-consuming, requires expensive reagents, and can yield variable results. Routine villous trophoblast isolation based on trypsin digestion of placental villi, followed by additional purification steps, appears to be costly and time-consuming (Kliman HJ et al., Endocrinology 1986;118:1567e82). Kilman et al. have shown that purifying villous trophoblasts using a Percoll gradient can achieve a purity of approximately 80% (Endocrinology 1986;118:1567e82; Clabault H et al., Methods Mol Biol. 2018;1710:219~231). Magnetic beads are also used to further purify choriotrophoblast cells from trypsin-treated placental explants (Douglas GC et al., J Immunol Methods 1989;119:259e68; Petroff MG et al., Methods Mol Med. 2006;121:203~217).Other research groups have successfully demonstrated the isolation of cytotrophoblast cells from mononucleated syncytia (Huppertz B et al., Lab Invest 1999;79:1687e702; Guilbert LJ et al., Placenta 2002;23:175e83; Tannetta DS et al., Placenta 2008;29:680e90). However, these methods still require secondary selective isolation techniques to achieve a reasonable level of purity from other placental cell types that are contaminants.

[0006] Therefore, in this field, there is still a need for streamlined and efficient methods to achieve reliable in vitro isolation of cells such as cytotrophoblast cells from placental tissue. [Overview of the project]

[0007] The problems described above and other problems are solved by the following inventions, however, it should be understood that not all embodiments of the inventions described herein solve the above problems. In some embodiments, it was unexpectedly discovered that the methods described herein achieve the secure in vitro isolation of cells such as cytotrophoblast cells from placental tissue.

[0008] In a first embodiment, a laboratory apparatus for isolating cells is provided, the apparatus comprising a hollow structure having a first open end and a second open end, wherein the first open end is coated with a lubricant and is configured to be positioned on the cells.

[0009] A second embodiment provides a method for isolating trophoblast cells, the method comprising: excising placental villous tissue from an isolated normal placenta; culturing the placental villous tissue in a culture medium under conditions suitable for trophoblast cell proliferation; and isolating the trophoblast cells using the above-described experimental apparatus to form isolated trophoblast cells.

[0010] A third embodiment provides a method for isolating trophoblast cells from placental tissue, the method comprising obtaining a detached normal placenta, collecting placental cotyledons from the detached normal placenta, excising placental villous tissue from the placental cotyledons, culturing the placental villous tissue in a culture medium, forming isolated colonies by placing the first open end of the experimental apparatus over colonies of trophoblast cells, and treating the isolated colonies of trophoblast cells with an enzyme solution.

[0011] The above provides a simplified summary that will give a basic understanding of some aspects of the claimed subject matter. This summary is not intended to provide a comprehensive overview, nor is it intended to identify key points or important elements, or to clarify the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed explanations that will be presented later.

[0012] Further features and advantages of this disclosure can be found in the following detailed description provided in conjunction with the drawings described below. [Brief explanation of the drawing]

[0013] [Figure 1] This shows cultured placental explants used for cell isolation in the trophoblast. [Figure 2]This is a schematic diagram illustrating a method for isolating trophotrophic cells from placental explants. Isolation of trophotrophic cells involves sectioning and arranging the villous explant on a petri dish, culturing and isolating selected cell colonies from the derivatives of the villous aggregates using a simple pipette barrel coated with Vaseline, followed by trypsin treatment and subculture, and confirmation of purity by cytokeratin 7 staining. [Figure 3A] As indicated by the white arrows, this is a fluorescence microscopy observation of living cells infected with HCMV-GFP, which expresses green fluorescent protein, in the peripheral placental tissue. [Figure 3B] As indicated by the white arrows, this is a fluorescence microscopy observation of contamination by red blood cells between living cells infected with HCMV-GFP, which expresses green fluorescent protein, in the periplacental tissue. [Figure 3C] As indicated by the white arrows, this shows fluorescence microscopy observations of living cells infected with HCMV-GFP, which expresses green fluorescent protein, and contamination by red blood cells in aggregated tissue. [Figure 3D] The images, indicated by the white arrows, show fluorescence microscopy observations of living cells of a villous tree structure infected with HCMV-GFP, which expresses green fluorescent protein. All images were acquired at 200x magnification using a Nikon TE2000S microscope equipped with a charge-coupled device (CCD) camera. [Figure 4A] This image shows placental nodes / cotyledons cultured in trophoblast cell culture medium 24 hours after excision and incision. [Figure 4B] This shows the proliferation (outgrowth) and early colony formation of villous cells after 8 days of culture. [Figure 4C] This shows proliferation of villous cells and subconfluent colony formation after 11 days of culture. [Figure 4D]The areas outlined with yellow dotted lines for clonal selection and proliferation show villous cell proliferation and confluent focal monolayers after 14 days of culture. [Figure 4E] This shows a villous tissue aggregate after 10 days of culture. [Figure 4F] This shows villous tissue aggregates and associated villous cell proliferation after 16 days of culture. [Figure 4G] Areas outlined with yellow dotted lines for clonal selection and proliferation show the proliferation of villous tissue aggregates and associated villous cells at higher magnification after 16 days of culture. [Figure 4H] The images show subculturing and culture of clonally selected cells that were positively stained for cytokeratin 7 by immunofluorescence staining 24 hours later. [Figure 4I] The images show subcultures and cultures of clonally selected cells that were positively stained for cytokeratin 7 by immunofluorescence staining after 48 hours. In the immunostained images, the nuclei were stained blue with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired at 200x magnification using a Nikon TE2000S microscope equipped with a charge-coupled device (CCD) camera. [Figure 5A] The image shows cells stained by immunofluorescence with an antibody against cytokeratin 7. The nuclei were stained blue with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired at 200x magnification using a Nikon TE2000S microscope equipped with a charge-coupled device (CCD) camera. [Figure 5B] The image shows cells stained by immunofluorescence with an antibody against cytokeratin 7. The nuclei were stained blue with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired at 200x magnification using a Nikon TE2000S microscope equipped with a charge-coupled device (CCD) camera. [Figure 5C]Cells stained by immunofluorescence staining with an antibody against cytokeratin 7 are shown. The nuclei were stained blue with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired at a magnification of 200-fold using a Nikon TE2000S microscope equipped with a charge-coupled device (CCD) camera. [Figure 5D] Cells stained by immunofluorescence staining with an antibody against cytokeratin 7 are shown. The nuclei were stained blue with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired at a magnification of 200-fold using a Nikon TE2000S microscope equipped with a charge-coupled device (CCD) camera. [Figure 6A] Cells treated with forskolin show high levels of hCG determined by IHC staining. [Figure 6B] Cells treated with forskolin show high levels of hCG determined by IHC staining. [Figure 6C] Cells treated with forskolin show high levels of hCG determined by IHC staining. [Figure 6D] Cells treated with forskolin show high levels of hCG determined by IHC staining.

Mode for Carrying Out the Invention

[0014] The drawings are not to scale and are intended to depict only typical aspects of the invention and should not, therefore, be regarded as limiting the scope of the disclosure.

[0015] A. Definitions Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as generally understood by those skilled in the art. Furthermore, terms as defined in commonly used dictionaries should be interpreted to have the same meaning as their meaning in the context of the specification, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in the specification. Well-known functions or structures may not be described in detail for the sake of brevity or clarity.

[0016] The terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context explicitly indicates otherwise.

[0017] In this specification, terms such as “first” and “second” are used to describe various features or elements, but these terms should not be used to limit those features or elements. These terms are used solely to distinguish one feature or element from another. Accordingly, without departing from the teachings of this disclosure, a first feature or element described below may be referred to as a second feature or element, and similarly, a second feature or element described below may be referred to as a first feature or element.

[0018] The term "consisting essentially of" means that, in addition to the enumerated elements, the claimed may also include other elements (such as steps, structures, ingredients, components, etc.) that do not adversely affect the operability of the claimed for the intended purposes described in this disclosure. The term excludes other elements that adversely affect the operability of the claimed for the intended purposes, as described in this disclosure, even if such other elements may enhance the operability of the claimed for other purposes.

[0019] The terms “about” and “approximately” generally refer to an acceptable range of error or variation in a measured quantity, taking into account the characteristics or precision of the measurement. A typical, exemplary range of error or variation is within 20 percent (%) of a given value or range of values, preferably within 10 percent, and more preferably within 5 percent. In biological systems, the term “about” refers to the standard deviation of the acceptable error, preferably no more than twice the given value.

[0020] The following description references several external documents to enable readers to understand and use the content described herein. Nothing included herein should be construed as an "admission" of prior art. The applicant expressly reserves the right, where appropriate, to prove that such documents referenced herein do not constitute prior art under applicable legal provisions.

[0021] B. Isolation method This disclosure provides methods for isolating cell populations from tissue. In some embodiments, this disclosure provides methods for isolating cytotrophoblast cells from placental tissue. Specifically, the methods disclosed herein enable routine culture and purification of choriotrophoblast cells from placental tissue explants in some embodiments. The methods described herein achieve robust in vitro isolation of purified cells.

[0022] In one embodiment, the method of the present disclosure utilizes placental tissue to isolate trophoblast cells, such as cytotrophoblast cells. As used herein, the term “trophoblast” refers to epithelial cells derived from the placenta of a mammalian embryo or fetus. Trophoblast cells are typically in contact with the uterine wall. Placental tissue contains three types of trophoblast cells: villous cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts. Therefore, as used herein, the term “trophoblast” encompasses any of these cells. Villous cytotrophoblasts are specialized placental epithelial cells that differentiate, proliferate, and further invade the uterine wall to form villi. Cytotrophoblast cells present in anchoring villi may fuse to form a syncytiotrophoblast layer or a column of extravillous trophoblast cells.

[0023] In this embodiment, the method includes obtaining a detached normal placenta. As used herein, “detached normal placenta” refers to a placenta separated from a healthy mother after birth. By using a detached normal placenta, the method of this disclosure eliminates the need to perform invasive laparoscopic surgery during pregnancy. The detached normal placenta needs to be stored quickly in a sterile environment.

[0024] In some embodiments, the method of the present disclosure involves culturing chorionic trophoblast cells from placental cotyledons. As used herein, “placental cotyledons” refers to the circular structures within the placenta that contain chorionic villi. In this embodiment, the method includes harvesting placental cotyledons from a detached normal placenta. Harvesting placental cotyledons can be carried out by any conventional anatomical method known in the art. In one embodiment, placental cotyledons can be excised from a detached normal placenta using any known surgical instrument, such as a scalpel and / or surgical scissors. In one embodiment, after excising placental cotyledons from a detached normal placenta, the placental cotyledons may be stored on ice for further use.

[0025] In one embodiment, placental villous tissue is collected from a placental lobe. Prior to collection, the placental lobe may be washed with a solution such as phosphate-buffered saline (PBS) or antibiotic-containing PBS (e.g., penicillin or streptomycin-containing PBS). The placental lobe may be washed two or more times; for example, it may be washed three times. The washing step can remove contaminants (e.g., blood) and hydrate the tissue. Extraction of placental villous tissue can be carried out by any conventional anatomical method known in the art. In one embodiment, the decidual layer may be removed to expose the placental villous tissue. The placental villous tissue can then be collected by finely dissecting the placental lobe using known surgical instruments such as a scalpel and / or surgical scissors. Placental villous tissue explants may be sectioned and placed in a sterile container such as a petri dish. As used herein, “explant” refers to a specimen obtained from any part of a living organism.

[0026] By sectioning the placental villous tissue explants and placing them in containers (such as Petri dishes or plates), culture media such as trophoblast cell culture medium can be added to each container. In some embodiments, the culture medium is a medium containing fetal bovine serum (FBS) and antibiotics. For example, the culture medium may contain 10 percent FBS and 1 percent antibiotics (e.g., penicillin or streptomycin). In one embodiment, the culture medium is added to the tissue explant in an amount of about 5 mL to about 30 mL. In another embodiment, the culture medium is added to the tissue explant in an amount of about 10 mL to about 20 mL. In yet another embodiment, the culture medium is added to the tissue explant in an amount of about 10 mL.

[0027] In some embodiments, the method of the present disclosure includes incubating a culture of placental villous tissue explants to promote the growth of trophoblast cell explants. The incubation of the culture may be about 1 to about 20 days. In another embodiment, the incubation of the culture may be about 1 to about 16 days. In yet another embodiment, the incubation of the culture may be about 3 to about 10 days. In yet another embodiment, the incubation of the culture may be about 16 days. In some embodiments, the culture is incubated under conventional culture conditions. For example, the culture can be incubated at a temperature of about 37°C. Furthermore, the culture medium may be replaced with fresh medium throughout the incubation period. In some embodiments, the medium may be replaced with fresh medium every 5 days, preferably every 3 days, and more preferably every 2 days.

[0028] In one embodiment, the proliferation of trophoblast cell explants is observed during the incubation period of colonies exhibiting trophoblast cell characteristics. In this embodiment, the proliferation of trophoblast cell explants is observed every three days, preferably every other day, and more preferably daily. For example, the proliferation of trophoblast cell explants can be examined by microscopic observation. In some embodiments, colonies of trophoblast cell proliferation may be identified morphologically. For example, colonies having morphological features and growth rates consistent with trophoblast cell monolayers may be identified. Once colonies of trophoblast cell proliferation are identified, they may be marked for isolation.

[0029] In some embodiments, this method involves isolating trophoblast cells, such as cellular trophoblast cells, from any of the identified explant proliferations. Prior to extraction of trophoblast cell colonies, any culture medium present in the container (e.g., Petri dish or plate) may be removed. Once the culture medium is removed, the trophoblast cell colonies can be isolated. In some embodiments, it is possible to isolate trophoblast cell colonies using a laboratory apparatus having a hollow structure. This apparatus may include any hollow structure having a first open end and a second open end. In one embodiment, it is possible to isolate trophoblast cell colonies using a modified standard pipette. In this embodiment, the tip and valve portion of the standard pipette may be removed to expose the barrel portion of the pipette. Any standard pipette can be used as long as the tip and valve portion can be removed to expose the barrel portion of the pipette. The dimensions of the pipette may vary depending on the size of the trophoblast population to be isolated. In some embodiments, this method utilizes a 1 mL standard pipette. In another embodiment, a standard cloning cylinder may be used, as long as the structure has at least two open ends. Any suitable sterilizable cylinder may be used (e.g., one made of polymer, glass, or metal). In some embodiments, the method utilizes antigen-specific antibodies on magnetic beads to confirm the isolation of trophoblast cells after subculturing in a chamber slide by indirect immunofluorescence staining.

[0030] Prior to isolation, one end of the laboratory equipment, for example, one end of the exposed barrel portion of a pipette or one end of a cloning cylinder, may be coated with a lubricant to help seal the edge of the hollow structure during isolation. Suitable lubricants include, but are not limited to, petrolatum, mineral oil, grease, polyalphaolefin (PAO), synthetic esters, polyalkyl glycol (PAG), and combinations thereof. Hydrophobic markers such as the IMMEDGE hydrophobic barrier pen can be used for isolation. In one embodiment, one end of the laboratory equipment may be coated with petrolatum. In another embodiment, one end of the laboratory equipment may be coated with mineral oil. In yet another embodiment, one end of the laboratory equipment may be coated with grease. The coated end of the laboratory equipment may then be placed directly above the trophoblast cell colony to be isolated. Advantageously, coating the end of the laboratory equipment with a lubricant such as petrolatum helps seal the edge of its structure for local trypsin treatment and prevents cross-contamination by suspension cells or cells from distal colonies. A hydrophobic marking pen can be used to create a seal around the selected colonies for trypsin extraction.

[0031] After isolating the selected trophoblast cell population, the selected population may be treated with an enzyme. In one embodiment, the selected trophoblast cell population may be treated with a solution of trypsin. For example, the selected trophoblast cell population may be treated with a 0.05 percent trypsin-EDTA solution. In some embodiments, the enzyme solution may be added directly to the trophoblast cell population via an exposed end (e.g., a second open end) of the apparatus while it is placed over the trophoblast cell colonies. In other embodiments, the enzyme solution may be added to the trophoblast cell population after the population has been extracted.

[0032] In some embodiments, a population of isolated cells is formed. As used herein, the term “population of isolated cells” means a population of cells substantially separated from other cells in the tissue from which the cell population originates (e.g., placenta). In one embodiment, a population of isolated cytotrophoblast cells is formed. The isolated cytotrophoblast cells can then be subcultured and verified for identity and purity. In some embodiments, isolated cytotrophoblast cells can be verified by measuring the expression of trophoblast cell marker molecules such as cytokeratin 7, hCG, HLA-G, placental alkaline phosphatase, and hyaluronic acid, which are targeted by the monoclonal antibody NDOG1. In preferred embodiments, isolated cytotrophoblast cells can be identified by immunofluorescence staining with an epithelial intermediate filament antigenic biomarker (cytokeratin 7). Isolated cytotrophoblast cells can be identified by measuring the expression of mesenchymal labeling (e.g., vimentin).

[0033] In some embodiments, the identification or purity of isolated cytotrophoblasts can be verified by measuring hCG expression in isolated cytotrophoblasts after treatment with forskolin.

[0034] While the method of isolation of cytotrophoblast cells from placental tissue has been described herein, those skilled in the art will understand that the described method can be modified to selectively isolate cell populations from various different tissues in multiple organ systems. For example, the disclosed method may be modified to isolate any of the following cell populations: stem cells (embryonic and adult), fibroblasts, chondrocytes, osteoblasts, osteoclasts, osteocytes, Merkel cells, and Langerhans cells, mesangial cells, podocytes, hepatocytes, pericytes, endothelial cells, cardiomyocytes, astrocytes, epithelial cells, astrocytes, ophthalmoocytes, adipocytes, lung cells, microglia, adrenal cortical cells, smooth muscle cells, macrophages, urothelial cells, keratinocytes, melanocytes, Sertoli cells, hepatic stellate cells, renal cells, cardiac cells, other visceral cells, and tumor cells.

[0035] As described herein, the disclosed method has several advantages, for example, selective isolation of cellular trophoblast cells. In some embodiments, populations of cells isolated by the disclosed method may be used in an in vitro placenta-on-chip model to study the biology and function of placental cells, as well as physiological responses after exposure to toxins and infectious pathogens. In other embodiments, populations of cells isolated by the disclosed method may facilitate in vitro studies of trophoblast cell proliferation, differentiation, invasion, viral interactions, cytokinesis, immunology, cell differentiation, cell transformation, cell division, apoptosis, and cell transplantation. In yet another embodiment, populations of cells isolated by the disclosed method may facilitate the development of microfluidic placenta models, sources of progenitor cells for differentiation, culture of purified cell populations for therapeutic purposes, development of on-chip organs, and development of purified cell injections.

[0036] C. Examples Example 1: Isolation of purified chorionic trophoblastic cells from placental explants

[0037] The following is an exemplary method for isolating cytotrophoblast cells from human placental villous explants.

[0038] <Materials and Methods> [Placenta collection]

[0039] The placenta was obtained from a cesarean section with elective painless delivery after full-term pregnancy at Vanderbilt University Medical Center. This study was approved by the Vanderbilt University Institutional Ethics Committee.

[0040] [Explant culture] Nodules were collected from intact placentas under sterile conditions. The chorionic nodules were first immersed in complete trophic cell culture medium and then placed on ice. Before incision, the nodules were washed with an excess of PBS to reduce contamination by red blood cells and to hydrate the tissue. The chorionic nodules were sectioned with a scalpel and surgical scissors and placed in a dish.

[0041] Figure 1 shows the cultured placental explant used for trophotrophic cell isolation. The chorionic nodules / placental lobes shown in Figure 1 were excised from the placenta using a scalpel and surgical scissors in a laminar flow hood and transferred to ice in a 50 mL conical tube containing 30 mL of trophotrophic cell medium. Prior to incision and culture, the nodules were immersed in phosphate-buffered saline (PBS) (pH 7.4).

[0042] [Cytotrophoblast cell isolation] All procedures involved in the collection and transfer of the placenta and placental explants were performed under sterile conditions. The time from delivery to placental nodule isolation was less than one hour.

[0043] Figure 2 is a schematic diagram illustrating the method for isolating cytotrophoblast cells from placental explants. To isolate the cytotrophoblast cells, the placental nodule or lobe was washed three times with pH 7.4 phosphate-buffered saline (PBS) and excised with sterile surgical scissors. The decidual layer was removed to expose the villous tissue. The placental nodule / lobe was finely dissected with a No. 21 replaceable-blade scalpel, and individual 5 mm tissue explants were arranged spatially on a 100 × 20 mm plate and cultured in 10 mL of trophoblast cell medium per plate (10% fetal bovine serum, 1% penicillin / streptomycin; ScienCell, Carlsbad, California). (Aronoff DM et al., Am J Reprod Immunol. 2017 September; 78(3):10.1111 / aji.12728). Explant cultures were incubated at 37°C for 1 to 16 days, with fresh culture medium replaced every two days. 25 μg / mL of fungizone (Gibco, Life Technologies, Grand Island, New York) was added to the trophoblast cell medium.

[0044] Trophoblast cell explant proliferation was examined daily by microscopic observation. Trophoblast cell colonies were identified using an inverted microscope. Morphologically identified colonies were circled with felt-tip markers before extraction. Colony extraction was performed using the barrel of a sterile 1 mL plastic pipette tip, after the tip was removed with a scalpel. The culture medium was removed from the dish, the open barrel of the pipette tip was immersed in sterile Vaseline petrolatum, and the tip of the Vaseline-soaked pipette barrel was applied directly to the trophoblast cell colonies on the plate. A seal was created around the cell colonies with Vaseline petrolatum. As shown in Figure 4B, selected colonies were identified first in the early stages of colony formation. Since tissue homogenization may increase the risk of potential contamination by endothelial cells, the isolation method does not necessarily have to include tissue homogenization. Trypsin (0.05% trypsin / EDTA (Gibco, Life Technologies, Grand Island, New York)) was added to the open end of the pipette barrel and allowed to stand at 37°C for 10 minutes. Trypsin-treated trophoblast cells were added to a 4-well chamber slide containing trophoblast cell medium. Confluent cells were confirmed to be trophoblast cells by staining with cytokeratin 7 (Millipore, Bedford, Massachusetts), an epithelial interlayer antigen biomarker. As described above, several colonies were selected by isolation using the trypsin-pipette-petroleum jelly method.

[0045] [Cells and viruses] Recombinant HCMV-GFP viruses expressing green fluorescent protein were obtained. HCMV-GFP was cultured in human preputial fibroblasts at a multiple of infection (moi) of 01. Viral titers were determined by limiting dilution using a fluorescence focus assay. (Alcendor DJ et al., J Neuroinflammation May 18, 2012; 9:95). Infection with SBCMV clinical strains was performed at passage level 3 (Alcendor DJ et al., J Neuroinflammation May 18, 2012; 9:95; Wilkerson I et al., J Neuroinflammation January 9, 2015; 12:2).

[0046] [HCMV-GFP infection of placental tissue culture] Chortrophoblast cells exhibit high tolerance to HCMV lysis and replication. To determine whether viable cells capable of supporting HCMV replication were present, tissue cultures of villous nodules were infected with recombinant HCMV-GFP. Approximately 20 grams of finely chopped villous tissue was added to 30 mL of trophoblast cell medium in a 50 mL conical tube preheated to 37°C. The trophoblast cell tissue cultures were infected with moi 0.1 of recombinant HCMV-GFP virus and incubated at 37°C for 1 hour (Aronoff DM et al., Am J Reprod Immunol. 2017 Sept;78(3):10.1111 / aji.12728). One hour after infection, the medium was removed and fresh medium was added. The tissue cultures were then incubated at 37°C for 96 hours. Infected villous tissue was placed in a chamber slide and GFP expression was examined using a fluorescence microscope.

[0047] [Immunofluorescence] Chamber slide cultures containing subcultured trophoblast cells were washed twice with PBS (pH 7.4), air-dried, and fixed in anhydrous methanol at -20°C for 10 minutes. The cells were air-dried for 15 minutes, hydrated with Tris-buffered saline (pH 7.4) for 5 minutes, and then incubated separately for 1 hour with mouse monoclonal antibody against cytokeratin 7 (Santa Cruz Biotechnology, Dallas, Texas) diluted 1:50 in PBS (pH 7.4). After washing the cells three times with Tris saline, they were incubated at 37°C for 30 minutes with a combination of fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, West Grove, Pennsylvania) and a secondary donkey anti-mouse immunoglobulin G (IgG) antibody diluted 1:100 in PBS (Alcendor DJ et al., J Neuroinflammation May 18, 2012; 9:95; Wilkerson I et al., J Neuroinflammation January 9, 2015; 12:2). The cells were then washed three more times with Tris saline and mounted in Vectorshield mounting medium (Vector Laboratories, Burlingame, California) containing 1.5 μg / mL of 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was captured using a Nikon TE2000S fluorescence microscope (Nikon, Tokyo, Japan) equipped with a charge-coupled device (CCD) camera.

[0048] [Immunohistochemistry] In trophoblast cell medium with or without forskolin, 1 × 10 4 Primary trophoblast cells were cultured in chamber slides at a cell / well density for 72 hours. The cells were washed three times with PBS pH 7.4, air-dried at room temperature, and fixed with 100% methanol at -20°C for 30 minutes. Next, the cells were air-dried, hydrated with PBS, and immunohistochemistry (IHC) was performed using a mouse monoclonal antibody against human chorionic gonadotropin (hCG) as described above. DAB (3,3-diaminobenzidine) was used as a peroxidase substrate for chromogenesis. Positive trophoblast cells appeared brown.

[0049] [Forskolin treatment] Primary trophoblast cells were placed in trophoblast cell medium in a chamber slide, 1 × 10⁶ cells. 4 Cells were cultured at a cell / well density and treated with 25 μM forskolin (Sigma-Aldrich Inc.) for 72 hours. Mock-treated cells were given only culture medium.

[0050] <Result> [Explant culture] Nodules were collected from sterile, intact placentas. The villous nodules were first immersed in complete trophoblastic cell medium and then placed on ice. Before incision, the nodules were washed with excess PBS to reduce red blood cell contamination and hydrate the tissue (Figure 1). The villous nodules were sectioned with a scalpel and surgical scissors and placed in a dish. Fresh, preheated trophoblastic cell medium was added, and the explant cultures were incubated at 37°C for 1 to 16 days, changing the medium every two days.

[0051] [Support for HCMV-GFP replication through placental tissue culture] Figures 3A-3D show placental explant tissue cultures infected with HCMV-GFP. As described above, placental explant tissue cultures were infected with HCMV-GFP, and GFP fluorescence was examined 96 hours after infection. 96 hours after infection, GFP-positive cells were observed under a fluorescence microscope in peripheral tissues and isolated cells (Figures 3A, 3B, 3C) as well as in the internal villous tree structure (Figure 3D). Significant contamination by erythrocytes was also observed in all examined samples.

[0052] [Nodular proliferation of cytotrophoblast cells] Figures 4A–4I show the isolation of trophoblast cells from full-term placental villous excisions after 16 days of trypsin treatment and subculturing. On day 1, the explants appeared aggregated and partially fixed (Figure 4A). On day 8 of culture, small colonies of cells with characteristic trophoblast cell morphology were observed (Figure 4B). On day 11, significant proliferation was observed in these colonies that had remained subconfluent (Figure 4C). After 14 days, patches of confluent growth with trophoblast cell morphology were observed (Figure 4D). These cells were round or oval, or multipolar, elongated cell monolayers previously described as a "crazy pavement." (Pennington KA et al., J.Vis.Exp.(59), e3202, doi:10.3791 / 3202 (2012); Kolokol'tsova TD et al., Bull Exp Biol Med. February 2015;158 (4):532~6; Aboagye-Mathiesen G et al., Clin Diagn Lab Immunol. January 1996;3(1):14~22; Li L et al., Reprod Biol Endocrinol. July 9, 2015;13:71). Furthermore, aggregates of trophotrophoblast cells were observed on day 10 after culturing (Figure 4E). Significant cell elongation was observed from these villous trophotrophoblast cell aggregates on day 16 (Figure 4F). These cells possessed characteristics consistent with villous trophotrophoblast cells.

[0053] [Selective capture and subculturing of purified cytotrophoblast cell populations] The development of confluent minicolonies and subsequent subconfluent nodule proliferation of satellite cells (Figure 4D), as well as cell proliferation obtained from nodule aggregates (Figures 4F and 4G), were preferred for selective capture and subculturing. These colonies appeared more homogeneous and possessed morphological features and growth rates consistent with trophoblast cell monolayers.

[0054] [Verification of trophoblast cell isolation] After 48 hours of trophoblast cell isolation, the subconfluent cell population that had been subcultured was observed to be viable, adherent, and positively stained for the trophoblast cell antigenic biomarker cytokeratin 7 (Figures 4H and 4I).

[0055] Figures 5A-5D show immunofluorescence staining of purified trophoblast cells expressing cytokeratin-7. As shown in Figures 5A-5D, universal cytokeratin-7 staining of the purified cell trophoblast layer was observed under a fluorescence microscope after the cells reached confluence. Primary trophoblast cells were placed in trophoblast cell medium in a chamber slide at a rate of 1 × 10⁶ 4 Cells were cultured at a cell / well density and treated with and without forskolin for 72 hours.

[0056] Figures 6A–6D show that cells treated with forskolin have higher levels of hCG, as determined by IHC staining.

[0057] D. Conclusion It should be understood that any given element of the disclosed embodiments of the present invention may be embodied in a single structure, a single step, a single substance, or otherwise. Similarly, a given element of the disclosed embodiments may be embodied in multiple structures, steps, substances, or otherwise.

[0058] The foregoing description exemplifies and illustrates the processes, machines, manufactures, composition of matters, and other teachings of this disclosure. Furthermore, while this disclosure shows and describes only specific embodiments of the disclosed processes, machines, manufactures, composition of matters, and other teachings, it should be understood that, as stated above, the teachings of this disclosure can be used in a variety of other combinations, modifications, and environments, and can be changed or modified within the scope of the teachings expressed herein in accordance with the skill and / or knowledge of those skilled in the art. The embodiments described herein further describe known specific best modes for carrying out the processes, machines, manufactures, composition of matters, and other teachings of this disclosure, and it is intended that those skilled in the art can utilize the teachings of this disclosure in such or other embodiments, and with various modifications required by a particular use or application. Accordingly, the processes, machines, manufactures, composition of matters, and other teachings of this disclosure are not intended to limit the exact embodiments and examples disclosed herein. None of the section headings in this specification are provided solely to conform to the implication of 37 C. FR § 1.77, or otherwise solely to provide an organized sequence of headings. None of these headings are intended to limit or characterize the inventions described herein. None of these headings are intended to limit or characterize the inventions described herein.

Claims

1. A method for isolating trophoblast cells, The extraction of placental chorionic tissue explants from a separated, normal placenta. The placental villous tissue explant is cultured in a culture medium under conditions suitable for the proliferation of trophoblast cells, and Remove the tip and valve portion of the plastic pipette to expose the barrel portion of the pipette, wherein the barrel portion of the pipette has a first open end and a second open end. The trophoblast cells are isolated by using the barrel portion of the pipette and coating the first open end of the barrel portion with a lubricant, wherein the lubricant is selected from the group consisting of mineral oil, polyalphaolefin (PAO), synthetic ester, polyalkylene glycol (PAG), and combinations thereof, and A method comprising positioning the coated first open end of the barrel portion onto the trophoblast cells in order to form isolated trophoblast cells.

2. The method according to claim 1, wherein the extraction step is: To obtain a separated, normal placenta, Collecting placental lobes from the separated normal placenta, and A method further comprising excising a placental villous tissue explant from the placental lobe.

3. A method according to claim 1 or 2, further comprising treating the isolated trophoblast cells with an enzyme solution containing trypsin.

4. A method for isolating trophoblast cells from placental tissue, To obtain a separated, normal placenta, To collect placental lobes from the separated normal placenta, To excise the placental villous tissue explant from the placental lobe, The placental villous tissue explant is cultured in a culture medium under conditions suitable for the proliferation of trophoblast cells. Remove the tip and valve portion of the plastic pipette to expose the barrel portion of the pipette, wherein the barrel portion of the pipette has a first open end and a second open end. By using the barrel portion of the pipette and coating the first open end of the barrel portion with a lubricant, the colonies of trophoblast cells are isolated, wherein the lubricant is selected from the group consisting of mineral oil, polyalphaolefin (PAO), synthetic ester, polyalkylene glycol (PAG), and combinations thereof, and By positioning the coated first open end of the barrel portion on the colony of trophoblast cells, an isolated colony is formed, and A method comprising treating the isolated colonies of trophoblast cells with an enzyme solution.

5. A method according to any one of claims 1 to 4, wherein the excision step further comprises sectioning the placental villous tissue explant.

6. A method according to any one of claims 1 to 5, wherein the culture medium comprises fetal bovine serum (FBS) and an antibiotic.

7. A method according to claim 6, wherein the culture medium comprises a solution of FBS, penicillin, and streptomycin.

8. A method according to any one of claims 1 to 7, wherein the placental villous tissue explant is cultured in 5 mL to 30 mL of culture medium.

9. A method according to any one of claims 1 to 8, wherein the placental villous tissue explant is cultured in 10 mL of culture medium.

10. A method according to any one of claims 1 to 9, wherein the culturing step further comprises incubating the culture medium for 1 to 16 days.

11. A method according to any one of claims 1 to 10, wherein the culturing step further comprises incubating the culture medium for 16 days.

12. A method according to any one of claims 1 to 3, further comprising staining the isolated trophoblast cells with cytokeratin 7 for verification of identity and purity.

13. A method according to claim 3 or 4, wherein the enzyme solution comprises 0.05 percent trypsin-EDTA.

14. A method according to any one of claims 4 to 13, further comprising removing the culture medium prior to the arrangement step.

15. A method according to claim 3 or 4, wherein the isolated colonies of trophoblast cells are treated with the enzyme solution by adding the enzyme solution through the second open end of the barrel portion.

16. A method according to any one of claims 1 to 15, wherein the trophotrophoblast cells are cellular trophotrophoblast cells.

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