Search

JP-7855233-B2 - Methods for isolating cells from placental tissue

JP7855233B2JP 7855233 B2JP7855233 B2JP 7855233B2JP-7855233-B2

Inventors

  • アルセンドル ドナルド

Assignees

  • メハリー メディカル カレッジ

Dates

Publication Date
20260508
Application Date
20200924
Priority Date
20190924

Claims (16)

  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, where 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, where 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. The 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.

Description

(Description of federally funded research or development) This invention was developed with government support under grant number S21MD000104, awarded by the National Institutes of Health (NIH). The government has certain rights to the invention. "Government" as used herein refers to the Government of the United States. This disclosure relates, in general terms, to a method for isolating a population of cells from a tissue, and more specifically, to a method for isolating trophoblasts from placental tissue. The placenta is a fetal organ involved in nutrient and gas exchange between mother and fetus throughout pregnancy. Abnormal placental formation can lead to various pregnancy complications (e.g., miscarriage, pre-eclampsia, and intrauterine growth restriction), which increases the risk of developing serious disabilities later in life (e.g., cardiovascular disease and type 2 diabetes). For this reason, understanding the molecular mechanisms governing human placental formation and trophoblast cell lineage is important. 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. Modeling of the human placenta to deepen our understanding of placental biology in human development has led 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 in vitro. 2019;54:105-113; Blundell C et al., Adv Health Mater. 2018;7(2)). These microdevices are fabricated 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 trophoblast cells. 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 trophoblast cells in vivo. (Pattillo RA et al., Cancer Res. 28:1231-1236, 1968; Pattillo RA et al., Ann. N.Y. Acad. 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 inconsistent results. Routine villous trophoblast isolation based on trypsin digestion of placental villi, followed by additional purification steps, appears to be costly and laborious (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 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 constitute impurities. 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. 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 all of 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. In the first embodiment, a laboratory apparatus for isolating cells is provided, 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 over the cells. In a second embodiment, a method for isolating trophoblast cells is provided, which includes excising placental villous tissue from an isolated normal placenta, culturing the placental villous tissue in a culture medium under conditions