A Method for Preparing Estradiol

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1. People’s Republic of China National Intellectual Property Administration

2. Invention Patent Application

3. Publication Number: CN 104293873 A

4. Publication Date: January 21, 2015

5. Application Number: 201310297273.2

6. Application Date: July 16, 2013

7. Applicant: Huashan Hospital Affiliated to Fudan University
   Address: No. 12, Middle Wulumuqi Road, Jing’an District, Shanghai 200031

8. Inventors: Liu Chunfang, Lü Yuan, Ma Zhan

9. Patent Agency: Shanghai Yuan Yi Cheng Intellectual Property Agency (General Partnership) 31268
   Agent: Wu Guiqin

10. International Classification:
    C12P 33/00 (2006.01)

Claims: 1 page

Description: 5 pages

Drawings: 4 pages

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54. Invention Title:
    A Method for Preparing Estradiol

55. Abstract:
    The invention belongs to the field of biological cell engineering and relates to a method for preparing estradiol, specifically involving the use of induced mammalian cells cultured in vitro to produce estradiol. The method employs a cell population derived from mammalian cells through special induction in vitro, which, under specific culture conditions, continuously secretes estrogen. The system simulates the natural biosynthesis process in vivo, resulting in fewer by-products, simplicity, mild conditions, and minimal environmental harm. Compared to existing preparation methods, it offers irreplaceable advantages. The invention provides a novel pathway for obtaining estradiol by enabling somatic cells of mammals to produce estradiol after induction under specific conditions in vitro.

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Claims

1. A method for preparing estradiol, comprising the following steps:
   1. Primary culture of isolated mammalian cells;
   2. Inducing the mammalian cells to obtain induced mammalian cells;
   3. Detecting and analyzing the ability of the induced mammalian cells to produce estradiol;
   4. Isolating and purifying to obtain pure estradiol.
2. The method according to claim 1, wherein:
   • Step (1):
     The isolated mammalian cells are primarily cultured in DMEM¹ medium containing 15% fetal bovine serum under conditions of 37°C and 5% CO₂ until reaching 10⁵ cells per flask.
   • Step (2):
     MCA² is added to the medium at a final concentration of 1 µg/mL, and the MCA-containing medium is replaced twice weekly. After one week of MCA treatment, the cells are maintained under standard culture conditions (37°C, 5% CO₂).
   • Step (3):
     After 3 months of culture, germ cell-like cells appear, expressing markers such as Oct4, Nanos3, c-Kit, DAZL, and Vasa.
3. The method according to claim 2, wherein:
   Step (3): MCA-induced hBMDCs³ show high estradiol expression (E2 = 3243 ± 947 pmol/L, n=6), a 50-fold increase compared to untreated cells (E2 = 65 ± 14.8 pmol/L, n=6). Follicle-like structures form, and follicle development-related genes (e.g., GDF9) are expressed.
4. The method according to claim 1, wherein the induction methods include chemical induction, natural substance treatment, gene transfection, radiation induction, viral induction, or long-term passaging.
5. The method according to claim 1, wherein the induced cells proliferate and differentiate into pluripotent stem cell-like cells, granulosa cell-like cells, primordial germ cell-like cells, or oocyte-like cells, which further organize into follicle-like structures to produce estrogen.
6. The method according to claim 1, wherein the primary culture is performed using bone marrow flushing or adhesion methods.
7. The method according to claim 1, wherein the isolated mammalian cells are human bone marrow-derived cells (hBMDCs, Deposit No. CCTCC C2012173), mouse bone marrow-derived cells (mBMDCs), mouse embryonic fibroblasts, pig embryonic skin cells, or rat embryonic stem cells.

Description

Technical Field

[0001] The invention pertains to biological cell engineering and provides a method for preparing estradiol using induced mammalian cells cultured in vitro.

Background

[0002] The prior art discloses that there are three main physiologically existing estrogens in the human body: estradiol, estrone, and estriol. These are primarily generated by the ovaries, follicles, corpus luteum, and placental tissue during pregnancy, and are of great significance for maintaining female secondary sexual characteristics. Estrogen deficiency can lead to functional decline in multiple organs of the female body. Data shows that when women enter menopause, estrogen levels drop sharply. In the United States alone, the market value of estrogen replacement drugs for menopausal women has reached nearly 20 billion US dollars.

[0003] Currently, the most commonly used drugs for estrogen supplementation fall into two categories: one being estrone derived from horse urine, and the other being chemically synthesized estradiol. Equine-derived estrone was the earliest estrogen replacement therapy and has been widely used worldwide for decades, becoming virtually synonymous with estrogen itself. Its preparation and purification methods are now quite mature. However, research shows that the estrogen actually needed by the human body is estradiol, while the estrogen components in pregnant mare’s urine mainly consist of estrone, equilin, and equilenin. The mentioned estrone is a metabolite of estradiol, with biological activity less than one-tenth that of estradiol, and carries toxic side effects including increased cardiovascular disease risk, liver damage, and breast cancer. Due to these significant adverse effects, estrone is commonly regarded as a “bad estrogen.”

[0004] Estradiol is primarily sourced from chemical synthesis. However, chemically synthesized estradiol presents problems including lengthy synthetic pathways, numerous byproducts, and environmental pollution. The ecological issues caused by estrogen analogs discharged into the environment have become increasingly concerning. As steroid hormones, estrogens differ from protein hormones in that they cannot be produced through the conventional bioengineering pathway of “DNA→mRNA→protein”, and can only be synthesized intracellularly using cholesterol as a substrate through enzymatic systems.

Under physiological conditions, estrogens mainly originate from theca cells and granulosa cells of ovarian follicles. During follicular development, luteinizing hormone (LH) first stimulates theca cells to secrete testosterone, which is then converted to estradiol by granulosa cells under the stimulation of follicle-stimulating hormone (FSH), and further metabolized into other estrogen components. Estrogen production involves coordinated actions among multiple cell types and hormones. Due to the extremely complex regulatory networks between various cells and hormones, cell engineering approaches for estrogen production face significant difficulties. To date, there have been no reports on successful cell engineering-based preparation of estrogens.

[0005] The inventors of this application intend to provide a method for preparing estradiol through genetic engineering or cell engineering.

[0006] References related to the present invention:

1. Beral, V. (2007). Ovarian cancer and hormone replacement therapy in the Million Women Study. The Lancet, 369(9574), 1703–1710. doi: 10.1016/S0140-6736(07)60534-0
2. Scarabin, P.-Y., Hemker, H. C., Clément, C., Soisson, V., & Alhenc-Gelas, M. (2011). Increased thrombin generation among postmenopausal women using hormone therapy: Importance of the route of estrogen administration and progestogens. Menopause, 18(8), 873. doi: 10.1097/gme.0b013e31820eee88
3. Renoux, C., & Suissa, S. (2011). Hormone Therapy Administration in Postmenopausal Women and Risk of Stroke. Women’s Health, 7(3), 355–361. doi: 10.2217/WHE.11.28
4. Liu, C., Xu, S., Ma, Z., Zeng, Y., Chen, Z., & Lu, Y. (2011). Generation of pluripotent cancer-initiating cells from transformed bone marrow-derived cells. Cancer Letters, 303(2), 140–149. doi: 10.1016/j.canlet.2011.01.021
5. Nicholas, C. R., Chavez, S. L., Baker, V. L., & Reijo Pera, R. A. (2009). Instructing an Embryonic Stem Cell-Derived Oocyte Fate: Lessons from Endogenous Oogenesis. Endocrine Reviews, 30(3), 264–283. doi: 10.1210/er.2008-0034

Summary of the Invention

[0007] The objective of the present invention is to overcome the deficiencies and shortcomings of existing technologies by providing a method for preparing estradiol. Specifically, the invention relates to a method for preparing estradiol through genetic engineering or cell engineering, with particular emphasis on the use of induced mammalian cells cultured in vitro to produce estradiol. This method establishes an in vitro cell engineering system utilizing induced mammalian cells for the preparation of estradiol.

[0008] In the method described in the present invention, the cell population used is derived from mammalian cells through special induction methods, including compound treatment, gene introduction, natural substance induction, viral infection, radiation induction, and long-term in vitro culture. These methods induce the in vitro cultured mammalian cells to acquire the ability to produce estradiol. Since the secretion of estradiol in this system simulates the biosynthetic process in vivo, the resulting estrogen exhibits complete species homology with the original cells. The production of estradiol generates few byproducts under mild conditions and poses almost no harm to the environment.

[0009] Specifically, the method for preparing estradiol according to the present invention is characterized by comprising the following steps:

1. Primary culture of isolated mammalian cells using existing techniques;
2. Inducing treatment of said mammalian cells to obtain induced mammalian cells;
3. Detecting and analyzing the ability of said induced mammalian cells to produce estradiol;
4. Isolating and purifying according to existing methods to obtain pure estradiol.

[0010] More specifically, the method for preparing estradiol according to the present invention is characterized by comprising the following steps:

1. Isolated mammalian cells are subjected to primary culture preparation using existing techniques. The prepared cells are cultured in DMEM medium containing 15% fetal bovine serum under conditions of 37°C and 5% CO₂ until reaching 10⁵ cells per culture flask.

2. MCA is added to the culture medium at a final concentration of 1 μg/mL, and the MCA-containing medium is replaced twice weekly. After 1 week of MCA treatment, the cells are maintained under standard culture conditions at 37°C and 5% CO₂.

3. After culturing for 3 months, germ cell-like cells are observed in the culture. These germ cell-like cells express germ cell markers: Oct4, Nanos3, c-Kit, DAZL, and Vasa.

   In the present invention, sustained expression of estradiol is detected in the culture medium. Compared to untreated hBMDCs (E2 = 65 ± 14.8 pmol/L, n=6), MCA-induced hBMDCs (E2 = 3243 ± 947 pmol/L, n = 6) exhibit a 50-fold increase in estradiol concentration (p < 0.01), demonstrating significant differences. After subculture, estradiol concentration progressively increases with prolonged culture time, peaking at approximately 10 days (up to 4500 pmol/L), then gradually decreasing while maintaining relatively high levels.

   In vivo, estradiol is synthesized by ovarian follicles. In the present invention, follicle-like structures are observed in MCA-induced hBMDCs cultured in vitro. Oocyte development requires estradiol stimulation—without sufficient estradiol, oocytes can only grow to a maximum of 25 μm. In contrast, oocyte-like cells in MCA-induced hBMDCs reach 40–50 μm in size, and immunofluorescence confirms these large cells as oocytes.

   The present invention involves follicle-related genes, including key estradiol synthesis genes (STAR, p450arom, p450c17) and follicle development-related gene (GDF9), which are expressed in the culture.

   By optimizing culture conditions, the invention enhances estradiol production capacity while reducing serum concentration in the medium. These germ cell-like cells further develop into early follicle-like structures and oocyte-like cells.

4. The estradiol is isolated and purified using existing methods.

[0011] In the present invention, said cell lines can be prepared by inducing transformation of normal mammalian cells through methods including chemical induction, natural substance treatment, gene introduction, radiation induction, viral induction, or long-term passaging. The induced cells, when cultured under high cell density conditions, can spontaneously proliferate and differentiate into cell populations including pluripotent stem cell-like cells, granulosa cell-like cells, primordial germ cell-like cells, and oocyte-like cells. Continued culture of these cells can further self-organize to form follicle-like structures, with the produced estrogen secreted into the culture medium, which can then be enriched and purified using existing techniques to obtain estradiol.

[0012] The advantages of the method of the present invention include:
The invention prepares estradiol through cell engineering means, wherein the employed cell population is generated by special in vitro induction of mammalian cells. Said cell population can be cultured under specific in vitro conditions to continuously secrete estrogen. The estrogen secretion in this system completely simulates the biosynthetic process in vivo, with few byproducts generated during estradiol production, simple methodology, and mild conditions that pose minimal environmental harm. Compared with existing preparation methods, it possesses irreplaceable advantages.

[0013] The cell engineering method for estrogen production of the present invention enables mammalian somatic cells to acquire estradiol-producing capability after special in vitro induction conditions, providing a novel pathway for obtaining estradiol.

Figures

[0020]

Detailed Description of Embodiments

[0021] The implementation process of the present invention will be illustrated by the following non-limiting examples.

[0022] Example 1

Human bone marrow-derived cells were induced using the compound 3-methylcholanthrene (MCA) to generate and screen the cells described in the present invention. These cells were used to synthesize estradiol in vitro. The cells have been deposited at the China Center for Type Culture Collection under accession number CCTCC C2012173.

Preparation steps:

1. Human bone marrow-derived cells (hBMDCs, primarily composed of human bone marrow mesenchymal stem cells) were prepared by primary culture using existing techniques (in this example, bone marrow flushing and adherent culture methods were employed). The prepared cells were cultured in DMEM medium containing 15% fetal bovine serum under conditions of 37°C and 5% CO₂ until reaching 10⁵ cells per culture flask.
2. MCA was added to the culture medium at a final concentration of 1 μg/mL, and the MCA-containing medium was replaced twice weekly. After 1 week of MCA treatment, the cells were maintained under standard culture conditions (37°C, 5% CO₂).
3. After culturing for 3 months, germ cell-like cells were observed in the culture (as shown in Figure 1A). These germ cell-like cells expressed germ cell markers: Oct4, Nanos3, c-Kit, DAZL, and Vasa (as shown in Figure 1B).
4. Sustained expression of estradiol was detected in the culture medium. Compared to untreated hBMDCs (E2 = 65 ± 14.8 pmol/L, n = 6), MCA-induced hBMDCs (E2 = 3243 ± 947 pmol/L, n = 6) exhibited a 50-fold increase in estradiol concentration (p < 0.01), demonstrating significant differences (as shown in Figure 2). After subculture, estradiol concentration progressively increased with prolonged culture time, peaking at approximately 10 days (up to 4500 pmol/L), then gradually decreasing while maintaining relatively high levels (as shown in Figure 3).
5. In vivo, estradiol is synthesized by ovarian follicles. In this invention, follicle-like structures were observed in MCA-induced hBMDCs cultured in vitro (as shown in Figures 4A-E), providing cellular support for estrogen production. Oocyte development requires estradiol stimulation—without sufficient estradiol, oocytes can only grow to a maximum of 25 μm. In contrast, oocyte-like cells in MCA-induced hBMDCs reached 40–50 μm in size (as shown in Figures 4I-G), and immunofluorescence confirmed these large cells as oocytes (as shown in Figures 4H-M).
6. Follicle-related genes, including key estradiol synthesis genes (STAR, p450arom, p450c17) and follicle development-related gene (GDF9), were expressed in the culture (as shown in Figure 5), further supporting the estradiol synthesis capability of induced hBMDCs.
7. By optimizing culture conditions, estradiol production capacity was enhanced while reducing serum concentration in the medium (facilitating further isolation and purification of estradiol). These germ cell-like cells further developed into early follicle-like structures and oocyte-like cells.
8. Estradiol was isolated and purified using existing methods.

[0023] Example 2

Mouse bone marrow-derived cells (mBMDCs) were induced through long-term in vitro culture to generate and screen the cells described in the present invention. These cells were used to synthesize estradiol in vitro.

Specific steps:

1. Mouse bone marrow-derived cells (mBMDCs) were isolated and purified, then subjected to continuous subculture in vitro. After approximately 20 passages⁴ or 6 months of continuous culture, the induced mBMDCs acquired the ability to produce estradiol.
2. The induced cells produced estradiol at concentrations up to 5000 pmol/L.

[0024] Example 3

Mouse embryonic fibroblasts were induced by introducing pluripotency genes (Oct4, Sox2, Nanog, c-KIT) in vitro to generate and screen the cells described in the present invention. These cells were used to synthesize estradiol in vitro.

Steps:

1. Mouse embryonic fibroblasts were isolated and cultured.
2. Pluripotency genes (Oct4, Sox2, Nanog, c-KIT) were introduced via genetic transfection. The resulting cells were screened, and those capable of producing higher estradiol concentrations were selected and expanded.
3. The induced cells produced estradiol at concentrations of approximately 3000 pmol/L.

[0025] Example 4 Porcine embryonic skin cells were induced by X-ray irradiation to generate and screen the cells described in the present invention. These cells were used to synthesize estradiol in vitro.

Steps:

1. Porcine embryonic skin cells were isolated.
2. The isolated cells were treated with X-ray induction, and the resulting cells were screened to select those capable of producing higher concentrations of estradiol, followed by expansion culture.
3. The induced cells produced estradiol at concentrations up to approximately 2500 pmol/L.

[0026] Example 5

Rat embryonic stem cells were induced to differentiate, generating and screening the cells described in the present invention. These cells were used to synthesize estradiol in vitro.

Steps:

1. Rat embryonic stem cells were isolated.
2. The isolated rat embryonic stem cells were deprived of feeder cells and LIF factor to induce spontaneous differentiation or differentiation under retinoic acid treatment. The resulting cells were screened to select those capable of producing higher concentrations of estradiol, followed by expansion culture.
3. The induced cells produced estradiol at concentrations up to approximately 4000 pmol/L.

Endnotes

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1. DMEM (Dulbecco’s Modified Eagle Medium): A widely used cell culture medium for mammalian cells, containing amino acids, glucose, vitamins, and salts.↩︎

2. MCA (3-Methylcholanthrene): A polycyclic aromatic hydrocarbon and potent carcinogen often used in research to induce cell transformation or differentiation. Here it acts as an inducer to reprogram mammalian cells (e.g., hBMDCs) into germ cell-like cells.↩︎

3. hBMDCs: Human bone marrow-derived cells are a pool of pluripotent stem and progenitor cells that include, among others, hematopoietic stem cells, mesenchymal stromal cells, and endothelial progenitor cells which secrete a variety of growth factors, cytokines, exosomes, and microvesicles. See doi: 10.1007/s00441-007-0509-0, and wikipedia.↩︎

4. Serial passage:

   Cells are split and transferred to fresh medium at 70–80% confluence (typically every 3-7 days).

   • 0% Confluence: No cells attached (empty flask).
   • 50% Confluence: Half the surface covered by cells.
   • 70–80% Confluence: Cells are nearly touching but not overcrowded (optimal for passaging, cells in their logarithmic growth phase, prevents contact inhibition).
   • 100% Confluence: Cells fully cover the surface, risking stress and differentiation.

   “Split and Transfer” Steps:

   1. Remove Old Medium: Aspirate the spent culture medium (contains waste products).
   2. Wash Cells: Gently rinse with PBS (phosphate buffered saline) to remove dead cells/debris.
   3. Detach Cells: Add a digestive enzyme (e.g., trypsin) to lift adherent cells from the flask surface.
   ↩︎