Methods and devices for generating oocytes with improved oocyte quality for in vitro fertilization procedures using non-invasive cell transfer

Abstract

The present invention relates to a method and device for generating mature oocytes from immature oocytes and improving the quality of oocytes to improve the success rate of assisted reproductive technology (ART). The method comprises: using tunneling nanotube-forming cells and oocytes, wherein the tunneling nanotube-forming cells transfer autologous genomic materials, biomolecules and cellular components to the oocytes. The device of the present invention comprises a microfluidic device to improve the efficiency of transferring biomolecules and cellular components between the tunneling nanotube-forming cells and the oocytes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. patent application Ser. No. 62 / 967,672, filed on Jan. 30, 2020, the entire contents of which (including any tables, figures, or drawings) are incorporated herein by reference. Background of the Invention

[0003] In vitro fertilization (IVF) is a technique used in assisted reproductive technology to improve fertility. Mature oocytes and sperm are collected from donors, and then the oocytes are fertilized in the laboratory. The fertilized eggs are then implanted back into the donor's uterus. The collection of human oocytes requires controlled ovarian hyperstimulation and transvaginal oocyte extraction. Controlled ovarian hyperstimulation mainly involves the injection of gonadotropins (such as follicle-stimulating hormone), followed by the injection of human chorionic gonadotropin (hCG) 36 hours before the extraction of oocytes. Depending on various protocols, injections are usually supplemented with gonadotropin agonists or antagonists. In transvaginal oocyte extraction, a needle is inserted through the vaginal wall into the ovarian follicles under ultrasound guidance. A suction device attached to the needle collects follicular fluid and oocytes. The collected mature oocytes are then used for IVF treatment and fertilization. These procedures are needed to obtain a larger number of fertilized eggs for IVF and implantation. Due to the low pregnancy rate of IVF, more than two embryos are usually transferred in a single implantation. Excess embryos from IVF can also be cryopreserved for use in a second implantation attempt.

[0004] In current practice, in vivo matured oocytes are used only in IVF practice, and most medical institutions discard immature oocytes due to their low fertilization potential. This practice is of little significance for young women undergoing IVF treatment, because a larger number of mature oocytes can be retrieved in a single round of controlled ovarian hyperstimulation. However, when older women (defined as 35 years or older) undergo IVF treatment, the number of mature oocytes collected is greatly reduced, reducing the chance of successful pregnancy after IVF treatment. Some IVF programs utilize in vitro maturation (IVM) as an alternative treatment, but the low success rate inhibits it from becoming a routine procedure. In vitro maturation is a clinical procedure used to process immature germinal vesicle (GV) stage oocytes and meiosis I (MI) stage oocytes to develop into mature MII stage oocytes. Despite recent advances in IVM, the fertilization potential of IVM-matured oocytes remains low. Therefore, it is desirable to develop an improved in vitro maturation method to utilize GV and MI oocytes.

[0005] Replenishing the loss of cytoplasmic material in aging cells can reverse the effects of aging. In fact, it has been reported that the expression of certain biomolecules (such as RNA and proteins) in aging oocytes is reduced. In addition to biomolecules, the reduction in the number and function of cellular components also leads to adverse reproductive outcomes in aging oocytes. For example, mitochondria are the "powerhouse" of cells and are key determinants of successful fertilization of oocytes. It generates 90% of cellular energy in the form of ATP. Mitochondria are also important in other functions such as maintaining ion homeostasis and cell apoptosis. The energy-generating capacity of mitochondria is crucial in oocyte maturation and successful fertilization, because some major events including chromosome segregation and zygotic genome activation depend on energy. Therefore, high-quality oocytes contain optimal mitochondrial numbers and sufficient levels of ATP. Aging is a risk factor for infertility due to decreased mitochondrial function. Some studies have linked increased oxidative stress to maternal age, and oxidative stress leads to mitochondrial dysfunction. Oxidative stress is often associated with mutations within the mitochondrial genome, which are more vulnerable due to the lack of histones. Therefore, mitochondrial function is often impaired in oocytes of older mothers. Reduced mitochondrial function in oocytes can lead to meiotic arrest and aneuploidy, which will result in the failure of successful pregnancy.

[0006] Therefore, mitochondria are the main target for improving the quality of oocytes in assisted reproductive technology. The latest methods include pronuclear transfer and maternal spindle transfer. Both methods require donor oocytes and may cause mitochondrial genomic heterogeneity. It is also described to separate oogonial stem cell (OSC) source compositions (such as anuclear cytoplasm or isolated mitochondria), and then such materials are manually injected into oocytes. However, the existence of OSC is still a controversial topic. Therefore, there is an urgent need for a self-supplementation method characterized by the non-invasive nature of no oocytes and the use of verified cell sources to improve the quality of oocytes. The present invention provides a non-invasive method, which transfers mitochondria from adult stem cells to oocytes by tunneling nanotubes. Summary of the invention

[0007] The present invention describes a new technology for improving oocyte quality in vitro, which greatly improves multiple pregnancy outcomes, such as maturation rate, fertilization rate and pregnancy rate. The new technology involves non-invasive transfer of biomolecules and cellular components from tunneling nanotube-forming cells to immature or mature oocytes through tunneling nanotubes.

[0008] The present invention relates to a method for maturing oocytes in vitro, comprising: incubating immature oocytes with tunneling nanotube-forming cells in an oocyte maturation medium using a unique culture method.

[0009] In some embodiments, the culturing method comprises: using a culture medium comprising an extracellular matrix.

[0010] In some embodiments, the methods of the present invention enable the transfer of biomolecules and cellular components from a cell source to an oocyte via a tunneling nanotube between two cells, thereby increasing the biomolecule and mitochondrial content in the oocyte.

[0011] In a specific embodiment, the method of the present invention includes: overexpressing a biomolecule encoded by the Kinesin-1 heavy chain gene (kif5b) in a tunneling nanotube-forming cell to increase the number and quality of tunneling nanotubes formed between the cell source and the oocyte, thereby effectively transferring biomolecules and cellular components.

[0012] In some embodiments, the culture method of the present invention comprises: culturing oocytes in a hanging drop having a certain concentration of tunneling nanotube-forming cells.

[0013] In additional embodiments, the present invention provides microfluidic devices that enable alternative culture methods that allow for the transfer of maximal biomolecules and cellular components between donor cells and oocytes.

[0014] In some embodiments, the present invention provides a microfluidic device made of polydimethylsiloxane (PDMS), in which oocytes are cultured together with tunnel nanotube-forming cells. In specific embodiments, microfluidic devices of the present invention maximize the formation of tunnel nanotubes between cells by making tunnel nanotube-forming cells and oocytes approach closely. Advantageously, device of the present invention enables accurate positioning of oocytes and tunnel nanotube-forming cells by controlling input flow rate, thereby maximally transferring biomolecules and cellular components.

[0015] The present invention also provides a method and device for sorting oocytes from tunneling nanotube-forming cells according to size and cell characteristics, respectively. In addition, the present invention provides a method for in vitro maturation, which allows culturing multiple oocytes in one device and exposing the multiple oocytes to tunneling nanotube-forming cells, and the present invention provides a method for improving oocyte quality by using tunneling nanotube-forming cells in a microfluidic device. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a schematic diagram of a hanging drop setup comprising an inverted hanging drop on a culture dish, and a cross-sectional view of a hanging drop culture comprising approximately 20 μL of culture medium with oocytes ( 6 ) and tunneling nanotube-forming cells ( 3 ).

[0017] FIG. 2( a ) shows a top view of a microfluidic device of the present invention having optimized channels and microchannels for maximizing the formation of tunneling nanotubes.

[0018] FIG. 2( b ) shows a top view of an alternative microfluidic channel having channels and microchannels for maximizing the formation of tunneling nanotubes for multiple tunneling nanotube-forming reactions.

[0019] Figure 3 A top view of another alternative microfluidic device of the present invention is shown, having preferred channels and microchannels for maximizing the formation of tunneling nanotubes.

[0020] Figure 4 Alternative microfluidic channel layouts of the device of the present invention are shown, illustrating designs for oocyte capture and cell-cell interactions.

[0021] Fig. 5 (a) shows a series of z-stacked laser confocal microscopy images, which show that biomolecules and cellular components are transferred from mouse adult stem cells (mMSC) to mouse germinal vesicle (GV) oocytes through tunnel nanotubes (TNTs). Mitochondria in adult stem cells are labeled with mitochondrial tracers (mitotracker, mitochondrial tracker). Arrows show that mitochondria are being transferred into mouse oocytes through tunnel nanotubes.

[0022] Figure 5(b) shows two confocal microscopy images of mouse oocytes after transfer of biomolecules and cellular components from mouse adult stem cells (mMSCs). The LC3 autophagosome protein is labeled with mCherry fluorescent protein. The arrows show that LC3 has been transferred into the mouse oocyte via the tunneling nanotubes.

[0023] Figure 6 Two scanning electron microscope images of mouse adult stem cells (mMSCs), meiosis II mouse oocytes, and tunneling nanotube (TNT) structures are shown at high magnification.

[0024] Figure 7 Schematic representation of ATP levels in oocytes without (GV) and with transfer of biomolecules and cellular components (GV+mMSC) as well as cells treated with rotenone (R).

[0025] Figure 8 Shown is a schematic diagram outlining the maturation rates of oocytes without (GV) and with transfer of biomolecules and cellular components (GV+mMSC) as well as cells treated with rotenone (R).

[0026] FIG. 9( a ) shows a microscopic image of the microfluidic tunneling nanotube-forming device of the present invention taken immediately after injection of mouse GV oocytes and adult stem cells.

[0027] FIG. 9( b ) shows a microscopic image illustrating successful maturation of mouse MII oocytes after transfer of biomolecules and cellular components from mouse adult stem cells (mMSCs) through tunneling nanotubes (TNTs) in the microfluidic tunneling nanotube forming device of the present invention.

[0028] Fig.10 Schematic diagram showing the enhancement of the maturation rate of adult stem cells supplemented with extracellular matrix (GV(R)+MSC) before and after transfer of biomolecules and cellular components using the microfluidic device of the present invention.

[0029] Fig.11 Two scanning electron microscope images of mouse menstrual blood derived cells (MBDCs), mouse oocytes, and tunneling nanotube (TNT) structures are shown at high magnification.

[0030] Fig.12 Schematic diagram showing the use of tunneling nanotubes to form menstrual blood-derived cells (MBDCs) or adult stem cells (iPSC-MSCs) to enhance the maturation rate of rotenone-treated GVs. DETAILED DESCRIPTION

[0031] definition

[0032] Oocyte

[0033] Oocytes are female germ cells involved in reproduction. Primary oocytes are arrested in the diplotene stage of meiotic prophase I during the fetal period. After puberty, large amounts of luteinizing hormone stimulate the resumption of meiosis. Oocytes mature during each menstrual cycle. The extrusion of the first polar body indicates that the oocyte is mature and ready for fertilization.

[0034] The immature oocytes used in embodiments of the present invention are oocytes in the developmental stage, including but not limited to germinal vesicle (GV) stage oocytes, meiosis I (MI) stage oocytes and follicles. In clinical in vitro fertilization (IVF) treatment, immature oocytes may be extracted together with mature oocytes during controlled ovarian hyperstimulation (COS) and transvaginal oocyte retrieval. Oocytes can also be extracted from women who have not yet received controlled ovarian hyperstimulation.

[0035] For humans, COS regimens vary depending on the patient, medical provider, and medical institution, but generally include, but are not limited to, administration of exogenous gonadotropins and / or co-treatment with gonadotropin-releasing hormone (GnRH) agonists or antagonists. The methods, durations, and doses of gonadotropins, GnRH agonists, and GnRH antagonists are not determinants of the present invention and are well documented in the prior art. Gonadotropins used in COS include, but are not limited to, human menopausal gonadotropin (hMG); urinary system products with follicle-stimulating hormone (FSH) or luteinizing hormone (LH) activity; purified FSH (p-FSH) or highly purified FSH (hp-FSH); or various recombinant FSH (rFSH) and LH (rFSH / rLH) preparations. At the end of the IVF cycle, human chorionic gonadotropin (hCG) or GnRH agonists or other substitutes may be administered at a certain time interval before the extraction of oocytes. Oocytes collected in COS can be used in embodiments of the present invention.

[0036] Biomolecules and Cellular Components

[0037] Biomolecules and cellular components refer to molecules and ions present in cells and living organisms. Biomolecules and cellular components include nucleic acids, proteins, phospholipids, carbohydrates, ions, fluids or cellular components composed of more than one type of biomolecule. For example, mitochondria contain biomolecules including, but not limited to, mitochondrial DNA, RNA, phospholipid membranes, mitochondrial proteins, and carbohydrates. Biomolecules and cellular components can be synthesized endogenously by the cell or introduced exogenously from external sources; thus, they can be naturally occurring or modified. External sources include, but are not limited to, media supplementation, transfection, and endocytosis.

[0038] In some embodiments, biomolecules and organelles are transferred from one cell to another. These biomolecules and organelles include, but are not limited to, nucleic acids, proteins, and mitochondria. They can be transferred by various mechanisms, including, but not limited to, tunneling nanotubes. The biomolecules and organelles may or may not change the recipient cell.

[0039] Tunneling nanotube-forming cells

[0040] Tunneling nanotube-forming cells refer to cell types that are able to transfer mitochondria, biomolecules, and cellular components (including but not limited to peptides and hormones) to another cell. Tunneling nanotubes are structures composed of microtubules and microfilaments that protrude from one cell to another. Cells connected by tunneling nanotubes may not be of the same type. Tunneling nanotube-forming cells include but are not limited to: mammalian adult stem cells, induced pluripotent stem cells, and menstrual blood-derived cells.

[0041] Adult stem cells

[0042] Adult stem cells refer to undifferentiated cells present in mammals that have the potential to differentiate into different cell types while being able to self-renew. Examples include: hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, adipose stem cells, and skin stem cells. Under normal physiological conditions, adult stem cells are mostly dormant. Glycolytic metabolism is the key energy production pathway in adult stem cells, thereby protecting the cells from reactive oxygen species (ROS) and mitochondrial damage.

[0043] Induced Pluripotent Stem Cells

[0044] Induced pluripotent stem cells refer to a type of pluripotent stem cells that are reprogrammed from somatic cells by inducing genes or factors. Examples include genomic integration of one or more Yamanaka factors, including but not limited to: Oct4, Sox2, Klf4, and c-Myc.

[0045] Menstrual blood cells

[0046] Menstrual blood-derived cells refer to cell populations isolated from human menstrual blood. These cells can be extracted by attachment to culture plates and further selected by fluorescence activated cell sorting. Red blood cells can be lysed after collecting menstrual blood. Density gradient centrifugation can be used to collect cell populations that can form tunneling nanotubes.

[0047] Oocyte handling before culture

[0048] Immature oocytes are processed prior to culture. Some immature oocytes are surrounded by a cumulus, also known as a cumulus-oocyte complex. For example, oocytes can be denuded or separated from other cellular components by using hyaluronidase in phosphate-buffered saline or culture medium. Mechanical exfoliation can also be used to facilitate denudation.

[0049] Hanging drop culture

[0050] Hanging drop culture refers to 3D hanging drop culture, which includes a drop of culture medium with cells or tissues suspended from an inverted surface.

[0051] Small culture system

[0052] Small culture system refers to a micro-volume culture system with a total internal volume and culture medium of less than 20 μL.

[0053] Microfluidic devices

[0054] Microfluidic devices refer to submillimeter devices that provide precise fluid manipulation and controlled environments for cell culture and growth. Cells in microfluidic devices are less affected by the external environment, and the differences between batches are smaller. In addition, due to its submillimeter scale, microfluidic devices minimize the number of cells used in the intercellular contact setting. The microfluidic device of the present invention is a specific submillimeter device, characterized in that a specific arrangement of microchannels is suitable for maximizing the intercellular contact between tunnel nanotube-forming cells and oocytes.

[0055] The present invention provides methods and devices for generating fertilization-competent oocytes from immature and mature oocytes using non-invasive techniques.

[0056] The present invention also provides methods and devices for in vitro maturation of immature oocytes. Advantageously, the methods and devices of the present invention allow for the non-invasive transfer of biomolecules and cellular components (including but not limited to whole mitochondria) to immature and mature oocytes, and maintain the quality and function of oocytes by inhibiting oocyte death that is usually associated with conventional microinjection and electrofusion methods.

[0057] In some embodiments, the method of the present invention comprises: using non-invasive biomolecule transfer means to transfer biomolecules (including but not limited to nucleic acids, proteins, phospholipids, carbohydrates, ions and fluids) to oocytes. Biomolecules useful in the present invention can be synthesized endogenously by cells or introduced exogenously from external sources.

[0058] In some embodiments, the methods of the invention comprise transferring cellular components including, but not limited to, nuclei, ribosomes, endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, secretory vesicles, microfilaments, and microtubules.

[0059] In the method and apparatus of the present invention, the oocyte employed can be the oocyte of any mammalian organism, including but not limited to: mankind, mouse, rat, ape, chimpanzee, orangutan, monkey, dog, cat, guinea pig, hamster, rabbit, ferret, cattle, horse, goat and sheep. In a preferred embodiment, the oocyte derives from mankind. In another preferred embodiment, the oocyte and biomolecule and cellular component are from the same species. In another preferred embodiment, the biomolecule and cellular component are from the same individual.

[0060] Adult stem cells used in the methods of the present invention include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, adipose stem cells, and skin stem cells. In some embodiments, menstrual blood-derived cells are used as donor cells for biomolecules and cellular components and tunneling nanotube-forming cells in the methods of the present invention.

[0061] The adult stem cells used in the methods and devices of the present invention can be adult stem cells of any mammalian organism, including but not limited to: humans, mice, rats, apes, chimpanzees, orangutans, monkeys, dogs, cats, guinea pigs, hamsters, rabbits, ferrets, cattle, horses, goats and sheep. In a preferred embodiment, the adult stem cells are derived from humans. In another preferred embodiment, the adult stem cells and oocytes are from the same species. In another preferred embodiment, the adult stem cells, oocytes and biomolecules and cellular components are from the same species. In another preferred embodiment, the adult stem cells, oocytes and biomolecules and cellular components are from the same individual.

[0062] The menstrual blood-derived cells used in the methods and devices of the present invention can be menstrual blood-derived cells of any mammalian organism, including but not limited to: humans, mice, rats, apes, chimpanzees, orangutans, monkeys, dogs, cats, guinea pigs, hamsters, rabbits, ferrets, cattle, horses, goats and sheep. In a preferred embodiment, the menstrual blood-derived cells are derived from humans. In another preferred embodiment, the menstrual blood-derived cells and oocytes are from the same species. In another preferred embodiment, the menstrual blood-derived cells, oocytes, and biomolecules and cellular components are from the same species. In another preferred embodiment, the menstrual blood-derived cells, oocytes, and biomolecules and cellular components are from the same individual.

[0063] In some embodiments, the oocytes used in the methods of the present invention are obtained from a mammal after controlled ovarian stimulation. In some embodiments, controlled ovarian stimulation is not required, as immature oocytes can be collected and matured using the methods and devices of the present invention. In this case, the extraction of oocytes from a mammal can be accomplished without the administration of gonadotropins, GnRH agonists or antagonists. In some embodiments, only hCG can be administered prior to the extraction of oocytes.

[0064] In some embodiments, the oocyte used in the methods of the present invention is an immature oocyte extracted from a mammal during an IVF procedure and destined for discard due to its immaturity.

[0065] In some embodiments, adult stem cells and / or menstrual blood-derived cells are used as the source of biomolecules and cellular components that are transferred to an oocyte using the methods and devices of the present invention.

[0066] In a preferred embodiment, adult stem cells are used for autologous biomolecule and cell component transfer and improve oocyte quality. Adult stem cells that can be used for the method of the present invention can be separated from various sources (including but not limited to: peripheral blood, bone marrow, adipose tissue, dental pulp, umbilical cord blood, menstrual blood and placenta). Adult stem cells can also be derived from peripheral blood mononuclear cells.

[0067] In a preferred embodiment, peripheral blood is collected from females with autologous oocytes using test tubes containing anticoagulants (including but not limited to heparin and test tubes coated with EDTA). Peripheral blood is treated with red blood cell lysis buffer, and whole blood is divided into different parts using density gradients (including but not limited to: ficoll-paque and hitopaque gradients). After centrifugation, the mononuclear cell layer is transferred to a culture dish and cultured in a culture medium containing various components (including but not limited to: 10-20% fetal bovine serum, autologous serum, human serum albumin, synthetic serum substitutes and / or merged human platelet lysates). The culture medium is renewed every 2-3 days until cell fusion is reached.

[0068] In an alternative embodiment, bone marrow is extracted from the left iliac crest of a female with autologous oocytes. Cells collected from the bone marrow are cultured on a plate in a culture medium containing various components, including but not limited to: 10-20% fetal bovine serum, autologous serum, human serum albumin, synthetic serum replacement, and / or pooled human platelet lysate. The culture medium is refreshed every 2-3 days until confluence is achieved.

[0069] In other alternative embodiments, human subcutaneous adipose tissue is obtained from females with autologous oocytes by liposuction. Adipose tissue stem cells are separated from the adipose stromal vascular part by attachment to a culture plate. The culture medium comprising various components (including but not limited to: 10-20% autologous serum, human serum albumin, synthetic serum substitute, fetal bovine serum and / or merged human platelet lysate) is renewed every 2-3 days until cell fusion is reached.

[0070] Advantageously, adult stem cells isolated using the methods of the present invention can be cryopreserved in liquid nitrogen prior to transfer of autologous and / or allogeneic biomolecules and cellular components to oocytes. Cryopreserved stem cells are thawed and recultured in standard culture media and conditions.

[0071] In a preferred embodiment, the tunnel nanotube is formed into cells with spheroid injection. Spheroid is a multicellular cell aggregate formed via an extracellular matrix, and the extracellular matrix is ​​combined by integrin to connect individual cells together. Compared with the single cell suspension of the same type, the cell spheroid generally has a larger surface area. Larger surface area promotes the contact between the tunnel nanotube and the oocyte. In addition, the tunnel nanotube can be formed between the cells of the spheroid, therefore, the success rate of forming the tunnel nanotube between the tunnel nanotube and the oocyte is increased, thereby improving the oocyte quality.

[0072] In some embodiments, using the method of the present invention, before adding one or more oocytes to improve the quality of oocytes, an extracellular matrix (including but not limited to collagen IV) is added to the tunnel nanotube-forming cell culture. The culture method varies between different types of tunnel nanotube-forming cells according to the specific cell type. In some embodiments, an extracellular matrix useful in the cell culture of adult stem cells and / or menstrual blood-derived cells is used. The technician can select an extracellular matrix suitable for the corresponding adult stem cells and / or menstrual blood-derived cells. In some embodiments, before being injected into the device of the present invention, the cells are digested with trypsin and cultured in a culture medium containing an extracellular matrix. The duration of the culture varies and will result in different spheroid shapes and sizes. In some embodiments, tunnel nanotube-forming cells are cultured in a culture medium including 1 mg / mL collagen IV for 20 minutes. Typically, the concentration of the extracellular matrix can be inversely proportional to the culture time. For example, the concentration of collagen IV can be a low amount of about 1 μg / mL to a high amount of about 100 mg / mL. In some embodiments, the concentration of the extracellular matrix can be about 2 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 50 μg / mL, about 100 μg / mL, about 200 μg / mL, about 500 μg / mL, about 1 mg / mL, about 2 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 50 mg / mL, to about 100 mg / mL.

[0073] The culture time of tunneling nanotube forming cells in the extracellular matrix can be as low as 5 minutes to as high as about 1 week. In some embodiments, the culture time can be about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 5 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 1 day, about 1.5 days, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days to about 1 week.

[0074] In some embodiments, adult stem cells and / or menstrual blood-derived cells of the present invention are transfected with a vector carrying the KIF5B gene. Transfer of the KIF5B gene to these cells results in overexpression of the KIF5B protein, which is involved in the distribution and transport of biomolecules and cellular components. Advantageously, overexpression of the KIF5B protein in adult stem cells and menstrual blood cells results in the formation of tunnel nanotubes in adult stem cells and menstrual blood-derived cells. Similarly, overexpression of the KIF5B protein in adult stem cells and menstrual blood cells increases the number of tunnel nanotubes formed by adult stem cells and menstrual blood-derived cells.

[0075] In an alternative embodiment, by increasing the expression of KIF5B protein in adult stem cells and menstrual blood-derived cells, the number of biomolecules and cellular components (including but not limited to mitochondria transferred to oocytes) is increased. Advantageously, the quality of oocytes after the transfer of biomolecules and cellular components enhanced after KIF5B overexpression is significantly improved. In some embodiments, KIF5B protein is overexpressed by transfecting a plasmid containing an expression vector, and the expression vector includes the sequence of the KIF5B gene. The gene encoding KIF5B protein can be from any mammal and non-mammalian organism. In a preferred embodiment, the KIF5B gene is a mammalian gene. In another preferred embodiment, the KIF5B gene is a human gene. The cloning and transfection methods of genes recorded in the art and the overexpression methods of proteins can be used in the methods of the present invention, and these methods are not described in detail herein. In some embodiments, KIF5B protein is supplemented to adult stem cells using microinjection or other protein injection techniques.

[0076] In some embodiments, methods and devices for generating tunneling nanotube-forming cells are provided. In specific embodiments, culture conditions and culture devices are provided for inducing adult stem cells and / or menstrual blood-derived cells to become tunneling nanotube-forming cells. In preferred embodiments, the generated tunneling nanotube-forming cells effectively transfer biomolecules and cellular components to oocytes.

[0077] In a specific embodiment, immature oocytes are cultivated together with the cumulus cells that surround each oocyte, also referred to as cumulus-oocyte complexes. In some embodiments, oocytes are stripped or separated from other cellular components by using hyaluronidase in phosphate buffered saline or culture medium. In some embodiments, mechanical stripping is used to promote stripping.

[0078] In some embodiments, the oocyte is cultured with the zona pellucida surrounding the oocyte. In other embodiments, the zona pellucida of the oocyte is removed using various methods, including but not limited to: mechanical stripping, acidic benchtop solution, laser treatment or treatment with pronase in culture medium. Advantageously, the removal of the zona pellucida can increase the number of biomolecules and cellular components transferred from the tunneling nanotube-forming cells to the oocyte through tighter cell-to-cell contact.

[0079] Culture conditions for oocytes and tunneling nanotube forming cells include, but are not limited to, culturing at 37°C in a humidified condition with an atmosphere of 95% CO2 and 5%, and include, but are not limited to, adding phosphate buffered saline around the culture, and optionally covering the culture with mineral oil.

[0080] The culture medium for oocyte and tunnel nanotube formation cells changes according to the donor and cell condition of cell. Single culture medium system or sequential culture medium system can be used for oocyte culture to improve oocyte quality. The component that oocyte culture medium of the present invention comprises includes but is not limited to: salt, essential amino acid and non-essential amino acid and energy source (such as glucose). The culture medium and culture condition that are usually used for oocyte culture also can be adopted in the method for the present invention.

[0081] In some embodiments, the culture medium of the present invention comprises one or more hormones. In a preferred embodiment, the hormones used in the method of the present invention promote the maturation of mammalian oocytes and increase their developmental capacity, and include but are not limited to: gonadotropins, such as follicle stimulating hormone (FSH), luteinizing hormone (LH), human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG). In some embodiments, hMG and hCG can replace FSH and LH, respectively. In a preferred embodiment, the concentration of hormones is adjusted according to cell quality, culture time, proliferation state and source species. The culture medium of the present invention also comprises serum supplements, including but not limited to: serum albumin, various growth factors essential for the growth of cultured cells. In some embodiments, fetal bovine serum (FBS) and serum from other individuals of the same species or serum from other species are used. In a preferred embodiment, non-animal substitutes are used in the method of the present invention, including but not limited to: recombinant human serum albumin or human patient's own serum. In another preferred embodiment, the culture medium used in the method of the present invention is supplemented with 10% heat-inactivated serum of human patients.

[0082] In some embodiments, the culture medium of the inventive method comprises extracellular matrix. Extracellular matrix (ECM) is a kind of non-cellular component, and it comprises polysaccharide, growth factor and protein.ECM serves as the structural support between cell and tissue, and is very important in cell-cell interaction and biochemical support.In a specific embodiment of the present invention, in cell culture, add ECM and can form an environment similar to the in vivo environment and promote cell growth.In some embodiments, extracellular matrix protein (including but not limited to: collagen, elastin, fibronectin and laminin) is added to culture medium.In some embodiments, two or more types of extracellular matrix protein are added to culture medium.In a preferred embodiment, low concentration of extracellular matrix protein is added to culture medium, and culture medium is maintained in liquid form rather than gel form to promote extraction simultaneously.In another preferred embodiment, in culture medium of the present invention, about 1.5mg / mL of type I collagen is added. In other embodiments, collagen, elastin, fibronectin and / or laminin is added to the cell culture medium at as low as 0.1 mg / ml to as high as 20 mg / mL, or at about 0.2 mg / mL to about 18 mg / mL; about 0.4 mg / mL to about 16 mg / mL; about 0.6 mg / mL to about 14 mg / mL; about 0.8 mg / mL to about 12 mg / mL; about 1.0 mg / mL to about 10 mg / mL; about 1.2 mg / mL to about 8 mg / mL; about 1.4 mg / mL to about 6 mg / mL; about 1.6 mg / mL to about 4 mg / mL; about 1.8 mg / mL to about 4 mg / mL; or about 2 mg / mL to about 3 mg / mL.

[0083] refer to Figure 1 , which illustrates the present invention, the present invention provides a hanging drop culture method to improve oocyte quality. For example, by using tunnel nanotubes to form cells, biomolecules and cell components are effectively transferred to oocytes, and oocyte quality is improved in hanging drop culture. The three-dimensional hanging drop culture of the present invention includes a culture medium droplet including cells and / or tissues suspended from an inverted surface. In some embodiments, oocytes (6) and tunnel nanotubes are formed in small droplets 2. The ratio of the surface area to volume of the culture medium is minimized by the hanging drop, thereby reducing the evaporation of the culture medium, and providing close proximity of the oocyte and the tunnel nanotube forming cells to effectively form tunnel nanotubes. Advantageously, the hanging drop allows the oocyte and the tunnel nanotube forming cells to gather in the middle of the droplet due to gravity, thereby maximizing the contact between the cells, and maximizing the transfer of biomolecules and cell components through the tunnel nanotubes to form nanotubes of adult stem cells and menstrual blood-derived cells.

[0084] In a preferred embodiment, the droplets of culture medium have a defined volume, include supplemented ECM proteins, serum and / or hormones, and are inverted on a plastic culture plate. For example, the volume of the droplets can be as low as about 0.1 μl / drop to as high as about 200 μl / drop. The droplet volume can also be about 0.2 μl, 0.3 μl, 0.4 μl, 0.5 μl, 0.6 μl, 0.7 μl, 0.8 μl, 0.9 μl and 1 μl to about 150 μl, 160 μl, 170 μl, 180 μl and 190 μl, and about 1.2 μl to about 145 μl, about 1.5 μl to about 140 μl, about 1.7 μl to about 135 μl, about 2 μl to about 130 μl, about 2.5 μl to about 125 μl, about 3 μl to about 120 μl, about 3.5 μl to about 115 μl, about 4 10μl to about 110μl, about 4.5μl to about 105μl, about 5μl to about 100μl, about 6μl to about 95μl, about 9μl to about 90μl, about 10μl to about 85μl, about 11μl to about 70μl, about 12μl to about 65μl, about 13μl to about 60μl, about 14μl to about 55μl, about 15μl to about 50μl, about 16μl to about 45μl, about 17μl to about 40μl, about 18μl to about 35μl, about 19μl to about 30μl, about 20μl to about 25μl. In a preferred embodiment, the droplet volume is about 10μl to about 60μl, about 15μl to about 50μl, or about 20μl to about 45μl. In another preferred embodiment, the droplet volume is about 10μl to about 40μl. In the most preferred embodiment, the droplet volume is 20 μl.

[0085] In another preferred embodiment, the droplet comprises one or more oocytes and about 1 to 50,000 tunneling nanotube-forming cells (TNFCs). In a more preferred embodiment, the droplet comprises 1,000 to 10,000 tunneling nanotube-forming cells; in an even more preferred embodiment, the droplet comprises 4,000 to 6,000 tunneling nanotube-forming cells.

[0086] In some embodiments, a droplet comprises one or more oocytes and has about 1 TNFC, 2 TNFC, 4 TNFC, 6 TNFC, 8 TNFC, 10 TNFC, 15 TNFC, 20 TNFC, 25 TNFC, 30 TNFC, 40 TNFC, 50 TNFC to about 10,000 TNFC, 15,000 TNFC, 20,000 TNFC, 25,000 TNFC, 30,000 TNFC, 35,000 TNFC, 40,000 TNFC, and 50,000 TNFC. In some embodiments, the droplets comprise one or more oocytes and about 500 to about 45,000 TNFCs, about 1,000 to about 40,000 TNFCs, about 1,500 to about 35,000 TNFCs, about 2,000 to about 30,000 TNFCs, about 2,500 to about 25,000 TNFCs, about 3,000 to about 20,000 TNFCs, about 3,500 to about 15,000 TNFCs, about 4,000 to about 10,000 TNFCs, about 4,500 to about 8,000 TNFCs, about 5,000 to about 6,000 TNFCs, or any number of TNFCs encompassed by and between said ranges.

[0087] In some embodiments, the hanging drop is inverted on the culture dish 1. In alternative embodiments, the culture dish is coated with a material to promote attachment of oocytes and TNFC, including but not limited to: extracellular matrix, cell attachment molecules, agar. In a preferred embodiment, the bottom of the culture dish 1 includes a defined volume of culture fluid to inhibit evaporation of the culture medium droplet, including but not limited to: phosphate buffered saline, 0.9% NaCl, or other cell-compatible liquids.

[0088] In some embodiments, the methods and devices of the present invention provide a small culture system. In some embodiments, the small culture system includes a micro-volume culture system, which in some embodiments includes an internal total volume of less than 20 μL and a culture medium. For example, the volume of a small cell culture system can be as low as about 0.1 μl to as high as about 100 μl. The volume can also be about 0.2 μl, 0.4 μl, 0.6 μl, 0.8 μl, 1 μl, 1.2 μl, 1.5 μl, 2 μl and 2.5 μl to about 95 μl, 90 μl, 85 μl, 80 μl and 75 μl, and about 0.2 μl to about 50 μl, about 0.4 μl to about 45 μl, about 0.6 μl to about 40 μl, about 0.8 μl to about 35 μl, about 1 μl to about 30 μl, about 1.2 μl to about 25 μl, about 1.4 μl to about 20 μl, about 1.4 μl to about 18 μl, About 1.6 μl to about 16 μl, about 1.8 μl to about 14 μl, about 2 μl to about 12 μl, about 2.2 μl to about 10 μl, about 2.4 μl to about 8 μl, about 2.5 μl to about 6 μl, and about 0.1 μl to about 5 μl, about 0.2 μl to about 4.5 μl, about 0.4 μl to about 4.5 μl, about 0.6 μl to about 4 μl, about 0.8 μl to about 3.5 μl, about 1 μl to about 3 μl, about 0.5 μl to about 1.5 μl, about 0.75 μl to about 1.25 μl, or about 1 μl.

[0089] In a preferred embodiment, the small culture volume is about 0.1 μl to about 5 μl. In another preferred embodiment, the small culture volume is about 1 μl.

[0090] Advantageously, the small culture system of the present invention allows close contact between the oocyte and one or more tunnel nanotube forming cells and minimizes the waste of culture medium. The small culture system of the present invention includes, but is not limited to, open systems, including but not limited to hanging drop-based and droplet-based cell culture systems; and closed systems, including but not limited to microfluidics.

[0091] In some embodiments, microfluidic device is provided to improve oocyte quality by forming cell transfer autologous biomolecules and cellular components from tunnel nanotubes.Microfluidic device generally refers to the submillimeter device that accurate fluid manipulation is provided.It can be used for biological and medical applications, and provides controlled environment for cell culture and growth.Advantageously, the cell in the microfluidic device of the present invention is less affected by the external environment, and the difference between batches is less.Because microfluidic device is normally micro-upgraded, therefore microfluidic device can minimize the quantity of cell and culture medium formed by tunnel nanotube used in the present invention.

[0092] In certain embodiments, the microfluidic systems of the present invention are made of various materials, including but not limited to polydimethylsiloxane (PDMS) and polymethylmethacrylate (PMMA). It is contemplated that any material suitable for making microfluidic devices can be used to make the microfluidic devices of the present invention.

[0093] In a preferred embodiment, a clean and transparent material is used to allow real-time monitoring of the period and condition of the oocyte in the embodiment. Standard photolithography techniques can be used, followed by casting and curing the polymer to manufacture the microfluidic device of the present invention. In a preferred embodiment, soft photolithography techniques are used, followed by casting and curing the PDMS prepolymer to manufacture the microfluidic device. SU-83025 can be used as a PDMS polymer by mixing a 10:1 base with a curing agent, and then curing at 60-70°C to manufacture a microfluidic chip. The microfluidic chip of the present invention can be attached to a coverslip using a 2:1 base to curing agent PDMS polymer, and preferably heated on a heating plate at 170°C. The curing temperature and the ratio of the base to the curing agent can be changed according to the needs of the user.

[0094] In a preferred embodiment, the microfluidic device of the present invention includes at least two openings connected to a closed space. In another preferred embodiment, the volume of the closed space in the microfluidic device is such that it preferably maintains enough culture medium for oocyte maturation, while being small enough to promote close cell-to-cell contact between oocyte and one or more tunnel nanotube-forming cells. Advantageously, the microfluidic device of the present invention allows accurate fluid manipulation and precise control of the environment to achieve optimal cell culture and growth. In addition, compared with the situation under conventional culture conditions, the cells in the microfluidic device are less affected by the external environment, and the batch-to-batch differences of the cultured cells are smaller. In some embodiments, the microfluidic device of the present invention has a closed space with a diameter of about 100 μm to about 2 mm and a height of about 10 μm to 200 μm. In some embodiments, the diameter of the enclosed space of the microfluidic device of the present invention is about 120 μm, about 140 μm, about 160 μm, about 180 μm, about 200 μm to about 1.6 mm, 1.8 mm, 2 mm, and the height is about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm to about 100 μm, about 120 μm, about 140 μm, 160 μm, about 180 μm or about 200 μm. In a preferred embodiment, the diameter of the enclosed space of the microfluidic device is about 100 μm to about 1.5 mm, or about 150 μm to about 1.2 mm, and the height is about 20 μm to about 180 μm, or 30 μm to about 160 μm. In another preferred embodiment, the diameter of the enclosed space of the microfluidic device is about 200 μm to about 1 mm, and the height is about 40 μm to about 150 μm.

[0095] In a preferred embodiment, the size of microfluidic device of the present invention is selected to adapt to the conditions and volume of used tunnel nanotube formation cells and oocytes. The shapes of the enclosed spaces are different. In some embodiments, the enclosed space is a cube, a cuboid, a cylinder, a cone, a tetrahedron, a triangular prism, a tetrahedron, an octahedron, a dodecahedron, an icosahedron, a hexagonal prism, a hexagonal pyramid, a pentagonal prism, a pentagonal pyramid, an octagonal prism or an ellipsoid. In a preferred embodiment, the enclosed space of microfluidic device of the present invention is a cylinder.

[0096] In some embodiments, the surface of the culture system can be coated with collagen. Collagen can be coated on a plate, a membrane, or any surface suitable for cell culture. The concentration or amount of collagen can vary depending on the cell state, the culture system surface, or other factors. The collagen coating can provide better attachment and growth for the cells, thereby improving the quality and maturity of the oocyte.

[0097] Referring to FIG. 2( a ), a top view of a preferred embodiment of a microfluidic tunnel nanotube forming device of the present invention is shown, which is used to transfer autologous biomolecules and cellular components between one or more oocytes and tunnel nanotube forming cells. The microfluidic tunnel nanotube forming device comprises: one or more channels (11); a microchannel system (13) comprising a plurality of microchannels; and at least one reaction chamber (12). In the channel (11), at least one oocyte can be placed and flowed to the reaction chamber (2) where the at least one oocyte is retained or placed.

[0098] Desirably, living cells (including but not limited to oocytes and tunnel nanotube forming cells) do not attach to the channels (11), reaction chambers (12) and / or microchannel systems (13) of the microfluidic device, and the materials used to make the microfluidic device include but are not limited to polydimethylsiloxane. Instead, living cells are placed or flow freely in the fluid or culture medium in the channels and microchannel systems. Such a microenvironment better mimics the in vivo environment. For example, the embryo migrates from the fallopian tube to the uterus in vivo without attaching to the sides of the fallopian tube. In some embodiments of the present invention, larger cells (such as oocytes) sink to the bottom of the channels and / or chambers of the device without attaching to the channels and / or chambers. Therefore, the movement of larger cells in the device may require fluid dynamics control so that the fluid flow moves the larger cells in the channels and / or chambers. Although the microfluidic device of the present invention can be made of any material commonly used to make cell culture devices, the preferred material for making the microfluidic device of the present invention is a material that does not allow cells to attach to its surface. However, if the tunnel nanotube forming cells attach to the material of the microfluidic device, trypsin or other cell dissociation methods known in the art can be used to remove cells from the surface.

[0099] In the embodiment of Figure 2(a), at least one inlet is located at the front end of the channel (11). In a preferred embodiment, the size of the inlet is generally larger than the diameter of the fluid manipulation device, such as, for example, the tip of a micropipette or the diameter of an adjustment handle. The size of the inlet depends on the material and test tube used to inject and manipulate cells and culture medium, and is configured to fit closely with the fluid manipulation device to allow fluids and cells to enter the channel (11) and prevent the fluids and cells from flowing out of the inlet due to lower resistance. The inlet of the channel can be in the same direction as the channel, or it can be tilted toward the top for ease of operation. In a preferred embodiment, in order to make it easier to use punching production, a vertical inlet with a 90-degree bend into the channel is used, and therefore, the shape of the inlet is generally cylindrical. In other embodiments, such as when the inlet is produced from the side, a "V" shape with a wider end away from the channel (11) is used to facilitate handling of culture medium with or without cells and adding it to the system.

[0100] In some embodiments, the channel (11) is connected from the inlet (optionally through pump 0) to the reaction chamber (12) and the microchannel system (13). The height and width of the channel vary according to the size of the cells used in the system. The size of the channel can be changed according to the user's requirements, for example, a narrow channel with a cell width can allow cells to pass through in a single file, so that tunnel nanotubes can be easily seen. In a preferred embodiment, the channel (11) is straight with smooth rounded corners to allow laminar flow. In other embodiments, the channel (11) has different shapes as long as laminar flow can be maintained. Longer and curved channels can be used to allow cells and oocytes to enter smoothly and provide culture medium storage. Constrictors and valves can also be added to the channel (11) to control the flow of the device. In a preferred embodiment, active flow is maintained for culture medium renewal. In some embodiments, the flow can be stopped to allow cells to interact and form tunnel nanotubes.

[0101] In some embodiments, the reaction chamber (12) allows cells (including but not limited to tunneling nanotube forming cells and oocytes) to interact and form tunneling nanotubes. In some embodiments, one or more reaction chambers can be designed to allow tunneling nanotubes to form simultaneously between cells.

[0102] In a preferred embodiment, the reaction chamber is circular to maximize the area used for tunneling nanotube formation, since many cells are circular when suspended. In another preferred embodiment, the size of the reaction chamber is not narrower than the channel (11), so that there is no resistance or only minimal resistance to prevent cells from entering and passing through. In a more preferred embodiment, the reaction chamber is designed as a continuation of the channel (11) without any separation.

[0103] In some embodiments, the reaction chamber is more than twice the diameter of the channel (11) to prevent the oocyte from escaping. In other embodiments, a valve is added between the channel (11) and the reaction chamber (12) to capture the oocyte and the tunnel nanotube-forming cells in the reaction chamber so that the small area maximizes the formation of tunnel nanotubes. The valve can also be used as a boundary between the channel (11) and the reaction chamber (12).

[0104] In some embodiments, the microchannel system (13) is located downstream of the reaction chamber (12) and upstream of the outlet. Advantageously, when the tunneling nanotube-forming cells and oocytes are inserted into the microfluidic device, the microchannel system can separate the cells based on their properties (such as size). In a preferred embodiment, when the sample includes at least one oocyte and a tunneling nanotube-forming cell, since the diameter of the MSC / tunneling nanotube-forming cell is about 10-40 μm and the diameter of the oocyte is about 80-100 μm, the microchannel can be adjusted to a diameter between the tunneling nanotube-forming cell and the oocyte, so that the tunneling nanotube-forming cell can pass through the microchannel and be extracted from the outlet, while the at least one oocyte is captured in the reaction chamber.

[0105] In some embodiments, the microfluidic device of the present invention comprises a single microchannel (13). In other embodiments, the microfluidic device of the present invention comprises a microchannel system (13), which comprises a plurality of microchannels for effective filtration. Advantageously, even in the presence of cell cluster formation, which would be captured in a narrow microchannel and completely block a single microchannel system, a multi-microchannel system allows filtration. In this embodiment, more microchannels are preferred so that even when cells block a microchannel, other microchannels can still be used to remove cells. An important factor is the total width of all microchannels (13). The sum of the widths of the microchannels (13) should not be less than the width of the channel (11), because if the sum of the widths of the microchannels (13) is much less than the width of the channel (11), a higher pressure will be generated in the upstream portion of the microfluid. In this case, the cells in the embodiment will be particularly exposed to mechanical stress conditions and will exert a cell stress response. In the worst case, the cells may rupture.

[0106] Furthermore, if the sum of the widths of all microchannels (13) is much smaller than the width of channel (11), larger cells may be squeezed through the microchannels (13) and removed from the outlet instead of being captured in the reaction chamber (12) due to increased pressure and faster flow rates in the reaction chamber (12) and the individual microchannels (13).

[0107] In a preferred embodiment, the flow rate in the microfluidic system of the present invention is slow, such as, for example, <10 μL / min, more desirably <5 μL / min, to prevent larger cells from being flushed out of the reaction chamber (12) through the microchannel (13).

[0108] If the cells used in the embodiment have similar sizes, cell characteristics other than cell size can be used to separate cells. For example, in some embodiments, microbeads and antibodies against different surface proteins are used to separate different types of cells. In some embodiments, magnetic beads are added to the channel through an outlet. Since the fluid containing magnetic beads is continuously pumped to the embodiment, the magnetic beads can be captured by a magnet placed outside the microchannel system (13) to keep the magnetic beads in place. In this embodiment, the magnetic side of the beads faces the magnet present outside the channel, and the antigen binding region faces the inside of the microchannel. In a preferred embodiment, when cells are injected, the cells of the target type are captured by cell-specific beads, thereby allowing other types of cells to pass through. Ultimately, the captured cells can be collected by removing the magnet and flushing the microchannel with a fluid.

[0109] In some embodiments, active flow can be used to renew nutrients and cells in microfluidic devices. Pump 0 is used to generate active flow in microfluidic devices. In some embodiments as shown in Figure 2 (a), the injection pump is connected to the outside of the microfluidic device, and the outlet of the pump is connected to the inlet of the microfluidic device. In other embodiments, a smaller pump adapted in the microfluidic device is used for easy processing. In the embodiment as shown in Figure 2 (a), a slower flow rate is expected, because the formation and transfer of tunnel nanotubes are processes that take minutes to hours, so a low flow rate can ensure that at least one oocyte is pushed away from the tunnel nanotube forming cell before forming a tunnel nanotube. In other embodiments, during the addition of culture medium and cells, the flow rate is changed to a different flow rate. Advantageously, the benefit of active flow is that, due to the micron width of the channel and microchannel in the microfluidic device, the amount of culture medium and nutrients can be relatively limited. However, due to the limited culture medium and space volume, the concentration of waste and toxic substances released by cells in the microfluidic device may increase faster than the concentration in other systems with a larger volume. Therefore, in a preferred embodiment, the culture medium is constantly renewed in the microfluidic device of the present invention to improve cell viability. In another embodiment, the culture medium is subjected to an active stream so that the stream allows the cells to move in the microfluidic device. Advantageously, the linear and rotational motion of the oocyte in the microfluidic system caused by this culture medium stream improves oocyte quality.

[0110] In some embodiments, such as the embodiment shown in Figure 2 (a), an optional filter (14) is used to retain cells in the microfluidic device circulation while removing spent culture medium from the microfluidic device. In some embodiments, the filter is located upstream of the outlet 15 in the microfluidic device so that cells captured inside the microfluidic device can be circulated back to the channel (11). In alternative embodiments, the filter is mounted on the outside of the microfluidic device for a larger surface area and higher filtration efficiency. In a preferred embodiment, the pores of the filter are smaller than the diameter of any cell used in the microfluidic device. For example, when tunneling nanotube-forming cells with a cell diameter of 20-50 μm are used in the system, a filter with a pore size of less than 15 μm will be used. In some embodiments, the channel can be blocked by setting a polydimethylsiloxane barrier with a size less than 15 μm in the channel. In other embodiments, alternative methods based on cell properties rather than size are used to separate and retain cells. For example, in some embodiments, cell shape, cell density, or magnetic properties of cells are used to separate and retain cells.

[0111] Referring to Fig. 2 (b), it shows a top view of another embodiment of the microfluidic tunnel nanotube forming device of the present invention. In a preferred embodiment, the channel (21) is connected to the reaction chamber (22) from the inlet. The width of the channel 21 is at least twice the diameter of the oocyte 6 plus the diameter of the tunnel nanotube forming cell. Therefore, in a preferred embodiment, the width of the channel (21) is at least 100 μm. In some embodiments, the width of the channel 11 is as low as 50 μm to as high as 500 μm, about 55 μm to about 450 μm, about 60 μm to about 400 μm, about 65 μm to about 350 μm, about 70 μm to about 300 μm, about 75 μm to about 250 μm, about 80 μm to about 200 μm, about 85 μm to about 150 μm, about 90 μm to about 125 μm. Advantageously, due to the width of the channel (21), the resistance to the injection of cells and medium is low, so that cell damage due to any forces applied during the injection of cells and medium does not occur.

[0112] In a preferred embodiment, the channel (21) includes a bend so that large oocytes 6 are trapped in the reaction chamber (22), which prevents the oocytes 6 from flowing back to the outlet. In a preferred embodiment, a right angle bend with smooth edges is provided to maintain flow while inhibiting cell trapping at the bend.

[0113] In some embodiments, the reaction chamber (22) is connected to at least one microchannel (23). In a preferred embodiment, the at least one microchannel (23) is narrower than the channel (21) and has a diameter between the size of larger cells (e.g., oocytes) and smaller cells (e.g., tunnel nanotube forming cells) used in the system, so that oocytes (6) can be captured in the reaction chamber (22) while smaller tunnel nanotube forming cells can pass through. Advantageously, the microfluidic device of the present invention allows for the sorting of two types of cells by size. In other embodiments, other cell characteristics and methods are utilized to sort and separate cells, including but not limited to: the presence of specific membrane proteins on the cell surface bound by specific antibodies and magnetic beads.

[0114] In some embodiments, a plurality of microchannels (23) are connected to the reaction chamber (22) from different angles and directions. In a preferred embodiment, at least three microchannels are connected to the reaction chamber (22).

[0115] In other embodiments, the microchannels (23) are connected to a central channel (24) which is connected to an outlet for removing cells and culture medium. In some embodiments, the microchannels are connected to the central channel (24) from different directions so that the microfluidic device of the present invention includes a bend and merges the microchannels (23) so that they are connected to the central channel (24). In a preferred embodiment, the bend is a smooth bend to prevent cells from clustering at the bend. In another preferred embodiment, the central channel (24) is a wide channel connected from at least one microchannel (23) to at least one outlet.

[0116] refer to Figure 3 , which shows an alternative embodiment of a microfluidic tunneling nanotube forming device used in a microfluidic cell co-culture system.

[0117] In the depicted embodiment, multiple inlets and channels (31a-b) are designed to connect the reaction chamber (12) from both sides. In a preferred embodiment, the inlets and channels are evenly distributed or equivalent. In other embodiments, other arrangements allow for easier operation with different distances between channels (31a), (31b) and outlets (35). In a preferred embodiment, oocytes and tunnel nanotube-forming cells are injected separately from the inlets upstream of the two channels (31a) and (31b). The two inlets are interchangeable, which means that there is no need to specifically select which cell type must be injected through which inlet. Channels (31a) and (31b) can be merged into one channel (31c) before being connected to the reaction chamber (32), or can be directly connected to the reaction chamber (32) without being merged into one channel together. The width of the merged channel (31c) is preferably greater than or equal to the width of the channel (31a) or (31b). Curved channels (31a) and (31b) allow for smooth injection of cells and oocytes.

[0118] In this embodiment, tunnel nanotube forming cells and oocytes are injected into the microfluidic tunnel nanotube forming device using an automatic pump. In a preferred embodiment, oocytes and tunnel nanotube forming cells are injected into the device simultaneously. Advantageously, a multichannel syringe pump assists by pumping the cell types simultaneously. After the oocytes and tunnel nanotube forming cells are transferred to two different syringes, the syringes are placed on a multichannel syringe pump and the cells are injected into the microfluidic tunnel nanotube forming device. Oocytes and tunnel nanotube forming cells will enter the reaction chamber 32 via channels 31a, 31b and 31c. In an alternative embodiment, oocytes and tunnel nanotube forming cells are injected separately. When cells are injected separately, a manual injection device or an automatic pump can be used. Preferably, oocytes are injected before injecting tunnel nanotube forming cells, so that during the injection of tunnel nanotube forming cells, oocytes will be captured in the reaction chamber 32, which can pass through the microchannel 33. When handled with caution, the reverse order can also work similarly.

[0119] refer to Figure 4 In some embodiments, additional designs of the microfluidic tunnel nanotube forming device used in a microfluidic cell co-culture system of the present invention are provided. In some embodiments, one or more cells are captured in at least one reaction chamber (42) while other cells pass through at least one channel (41). In some embodiments, a microchannel (43) connects the reaction chamber (42) to a downstream channel.

[0120] In some embodiments, at least one reaction chamber (42) is directly located at the bend of the channel, where one or more oocytes (6) from the same patient can be loaded into the reaction chamber by controlling the flow rate. In some embodiments, another batch of cells including tunnel nanotube-forming cells are then injected into the channel of the device. Advantageously, cells located near the reaction chamber (42) interact with the first batch of tunnel nanotube-forming cells and form tunnel nanotubes. In some embodiments, in order to remove the oocytes (6) captured in the reaction chamber (42), culture medium or fluid is injected from the outlet so that the fluid flows into the reaction chamber (42) through the microchannel (43) and the oocytes (6) are pushed out and collected at the entrance. Advantageously, this design allows a large number (theoretically hundreds or thousands) of reaction chambers (14) to be connected together at the same time to conduct tunnel nanotube formation research.

[0121] The invention is further illustrated by reference to the following non-limiting examples.

[0122] All patents, patent applications, provisional applications, and publications referenced or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0123] The following are examples illustrating procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0124] Example 1

[0125] Effects of tunneling nanotube-forming cells on mouse oocytes using a hanging drop culture system

[0126] The effects of transferring autologous biomolecules and cellular components to mouse oocytes using the hanging drop method during in vitro maturation were determined. C57 / BL6 mice aged 6 weeks or 12 months were perfused with PMSG (5 IU / female). Female mice were killed by cervical dislocation 48 hours later. Alcohol was sprayed into the abdomen of the mice and an incision was made to expose the abdominal cavity. The ovaries were dissected with a needle and cut into small pieces. Germinal cyst (GV) oocytes were selected and either control (medium only) or co-cultured with adult stem cells. Prior to co-culture, cumulus cells surrounding the oocytes were removed using 1 mg / μl hyaluronidase in M2 medium (Sigma-Aldrich, USA).

[0127] To simulate poor oocyte quality (such as by inhibiting mitochondrial respiratory function), oocytes were treated with rotenone at a concentration of 0.05 μM for 2 hours. Oocytes were collected after 24 hours of co-culture.

[0128] Adult stem cells were collected from adipose tissue before in vitro maturation. C57 / BL6 mice were killed by cervical dislocation. 5 mg of subcutaneous fat was dissected from mice. After being cut into small pieces, the subcutaneous fat was digested with type I collagenase (2 mg / mL in PBS, Thermo Fisher Scientific, USA) at a temperature of 37 ° C. The sample was then centrifuged at 400 g for 15 minutes, and the matrix vascular fraction was collected and cultured on a culture dish (Thermo Fisher Scientific, USA) in DMEM low glucose medium supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, USA) and 1% penicillin / streptomycin (Thermo Fisher Scientific, USA) until cell fusion was reached. The culture medium was replaced every 2-3 days. Before co-culture, the mitochondria of adult stem cells were labeled with a green mitochondrial tracer. In another preparation of adult stem cells, adult stem cells were transfected with a plasmid containing the LC3 autophagosome gene labeled with mcherry. The expression of LC3-mCherry in adult stem cells was confirmed by flow cytometry.

[0129] IVM culture medium is prepared using Alpha MEM without amino acids (except glutamine), vitamins and nucleosides. Newly prepared simplified Alpha MEM comprising 10% FBS and 0.2IU / mL FSH. Selected oocytes are washed 3 times in M2 culture medium, and cultured in the hanging drops consisting of 20 μl IVM culture medium (for treatment group and control group, respectively with and without 5000 suspended adult stem cells) at 37 ℃ in the humidified air containing 5% CO2. After co-cultivation for 24h, 0.25% trypsin (Life Technologies, the U.S.) is used to separate the MSC on the oocyte. Oocytes with polar bodies are recorded as mature oocytes.

[0130] result

[0131] To confirm the successful transfer of mitochondria from adult stem cells to at least one oocyte, the mitochondria of the adult stem cells were labeled with a green mitochondrial tracer before co-culture. The presence of the green mitochondrial tracer signal in the oocyte confirmed that the adult stem cells had endowed the oocyte with mitochondria from the adult stem cells through the tunnel nanotubes (Figure 5(a)). To confirm the transfer of proteins from adult stem cells to at least one oocyte, a plasmid containing autophagosomes labeled with fluorescent proteins was transfected into the adult stem cells before co-culture. The presence of the red mCherry signal in the oocyte confirmed that the adult stem cells had transferred proteins from the adult stem cells to the oocyte through the tunnel nanotubes (Figure 5(b)). Within 2 hours of culture, tunnel nanotubes were formed between the adult stem cells and the oocyte. The tunnel nanotubes penetrated the oocyte zona pellucida and were in close contact with the cytoplasm of the oocyte. Scanning electron microscopy also confirmed that the tunnel nanotubes were connected from the adult stem cells to the oocyte ( Figure 6 ).

[0132] To demonstrate the effects of mitochondrial, biomolecular, and cellular component transfer on improving the quality of oocytes with mitochondrial dysfunction, the internal ATP content of oocytes with and without mitochondrial transfer after germinal vesicle breakdown (GVBD) was investigated. The results showed that mouse MSCs (mMSCs) increased the ATP content of rotenone-treated oocytes (GV(R)) by 13.3% ( Figure 7 ). In addition, when mMSCs were treated with rotenone, the ATP content of GVs with mitochondrial dysfunction was significantly reduced to less than 0.1 pmol / oocyte.

[0133] To investigate how mitochondria transfer from adult stem cells to oocytes supports oocyte maturation with mitochondrial dysfunction, the maturation rates of oocytes with or without mitochondrial transfer were compared. The maturation rate of oocytes treated with rotenone was drastically reduced, but significantly increased by 80.4% ( Figure 8 ). These results suggest that mMSCs can support blastocyst rupture and GV(R) maturation through mitochondrial transfer and increased ATP content. However, MSCs with mitochondrial dysfunction did not support GV(R) maturation and significantly decreased the ratio by 56.7%.

[0134] To investigate how mitochondrial transfer improves oocyte quality in aged mice, oocytes from 12-month-old mice were used. Mitochondrial transfer from aged adult stem cells resulted in an increase in maturation and fertilization rates of 59.3% and 50.0%, respectively (Table 1). The number of embryos with ≥6 cells also increased by 50% after mitochondrial transfer. At the same time, the polar body fragmentation rate, a hallmark of poor oocyte quality, was reduced by 58.6% after autologous mitochondrial transfer.

[0135] Table 1

[0136]

[0137]

[0138] Example 2

[0139] Effects of overexpression of KIF5B in adult stem cells on mouse oocytes using a hanging drop culture system ring

[0140] The effects of autologous transfer of biomolecules and cellular components from adult stem cells overexpressing the KIF5B gene into mouse oocytes using the hanging drop method were determined. The KIF5B gene was cloned into an expression vector plasmid, and the plasmid was transfected into human adult stem cells.

[0141] C57 / BL6 mice were perfused with PMSG (5 IU / female). Female mice were killed by cervical dislocation 48 hours later. Alcohol was sprayed into the abdomen of the mouse, and an incision was made to expose the abdominal cavity. The ovaries were dissected with a needle and cut into small pieces. Germinal cyst (GV) oocytes were selected and controlled (medium only) or co-cultured with adult stem cells. Before co-culture, 1 mg / μl hyaluronidase was used to remove the cumulus cells around the oocytes in M2 culture medium (Sigma-Aldrich, USA). In order to simulate poor oocyte quality (such as the effects of inhibiting mitochondrial respiratory function), oocytes were treated with 0.05 μM concentration of rotenone for 2 hours. Oocytes were collected after 24 hours of co-culture.

[0142] IVM culture medium is prepared using Alpha MEM without amino acids (except glutamine), vitamins and nucleosides. Newly prepared simplified Alpha MEM comprising 10% FBS and 0.2IU / mL FSH. Selected oocytes are washed 3 times in M2 culture medium, and cultured in the hanging drops consisting of 20 μl IVM culture medium (for treatment group and control group, respectively with and without 5000 suspended adult stem cells) at 37 ℃ in the humidified air containing 5% CO2. After co-cultivation for 24h, 0.25% trypsin (Life Technologies, the U.S.) is used to separate the MSC on the oocyte. Oocytes with polar bodies are recorded as mature oocytes.

[0143] result

[0144] Overexpression of KIF5B in adult stem cells increased the maturation rate and mitochondrial function of rotenone-treated oocytes with mitochondrial dysfunction by 26.9% and 13.3%, respectively (Table 2). In addition, adult stem cells overexpressing KIF5B can rescue oocytes by increasing the survival rate of oocytes by 18.2%. These findings indicate that adult stem cells overexpressing KIF5B have a good improvement effect on oocytes with mitochondrial dysfunction.

[0145] Table 2

[0146]

[0147] Example 3

[0148] Effects of adult stem cells on mouse oocytes using the microfluidic device of the present invention

[0149] The effect of transferring autologous biomolecules and cellular components from adult stem cells to mouse oocytes was determined in vitro using the microfluidic device of the present invention. 6-week-old C57 / BL6 mice were perfused with PMSG (5IU / female). Female mice were killed by cervical dislocation 48 hours later. Alcohol was sprayed into the abdomen of the mouse, and an incision was made to expose the abdominal cavity. The ovaries were dissected with a needle and cut into small pieces. Germinal cyst (GV) oocytes were selected and controlled (only medium) or co-cultured with adult stem cells. Before co-cultivation, 85IU / ml hyaluronidase was used to remove the cumulus cells around the oocytes in M2 culture medium (Sigma-Aldrich, the United States). In order to simulate poor oocyte quality (such as by inhibiting mitochondrial respiratory function), oocytes were treated with rotenone at a concentration of 0.05μM for 2 hours.

[0150] Adult stem cells were collected from adipose tissue before in vitro maturation. C57 / BL6 mice were killed by cervical dislocation. 5 mg of subcutaneous fat was dissected from mice. After being cut into small pieces, subcutaneous fat was subjected to type I collagenase (2 mg / mL in PBS, Thermo Fisher Scientific, USA) digestion for 1 hour at 37 ° C. The sample was then centrifuged at 400 g for 15 minutes, and the matrix vascular part was collected and cultured in a culture dish (ThermoFisher Scientific, USA) in a DMEM low glucose medium supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, USA) and 1% penicillin / streptomycin (Thermo Fisher Scientific, USA) until fusion. The culture medium was replaced every 2-3 days.

[0151] The microfluidic device of the present invention including an inlet, a reaction chamber and an outlet was fabricated using SU-8 3025 PDMS polymer. The PDMS polymer was mixed with a 10:1 base to curing agent and then cured at 60-70° C. The microfluidic chip was attached to a cover glass using a 2:1 base to curing agent PDMS polymer and preferably heated on a hot plate at 170° C.

[0152] IVM culture medium was newly prepared using M2 culture medium containing type I collagen (1.5 mg / ml). Before use, the selected oocytes were randomized and washed 3 times in M2 culture medium. Adult stem cells were dissociated and counted from culture dishes. 20 μL of IVM culture medium with a concentration of 250 MSC / μL was prepared and injected into a microfluidic device with oocytes at a steady rate. The process was observed under a stereomicroscope. Once the oocyte entered the reaction chamber, the injection speed was slowed down. When the oocyte was surrounded by adult stem cells, the injection was stopped. The control group consisted of oocytes cultured alone in IVM culture medium in a microfluidic device.

[0153] After culturing in the microfluidic device for 24 hours or when oocytes matured to the M2 stage, IVM culture medium was injected from the inlet to remove MSCs from the microfluidic device. Once MSCs were removed from the microfluidic device, IVM culture medium was injected from the outlet to extract oocytes from the inlet. If MSCs were attached to oocytes and could not be removed, 0.25% trypsin (Life Technologies, catalog number 25200-056) was used to separate MSCs.

[0154] result

[0155] Microscopic images showed that the oocytes and adult stem cells were in close proximity and that the oocytes successfully matured from the GV to the MII stage (FIG. 9(a) and FIG. 9(b)). The bar graphs showed that by using autologous mitochondrial transfer from adult stem cells in the microfluidic device of the present invention, the maturation rate of the oocytes treated with rotenone increased by 20% compared to using IVM medium alone in the microfluidic device ( Fig.10 ).

[0156] Example 4

[0157] Effects of menstrual blood-derived cells on oocytes using the microfluidic device of the present invention

[0158] Menstrual blood was collected from a 25-year-old healthy female donor using a menstrual cup. 5 ml of menstrual blood was mixed with an equal amount of phosphate buffered saline (PBS) containing penicillin-streptomycin and 2 mM ethylenediaminetetraacetic acid (EDTA). Menstrual blood mononuclear cells were separated using Ficoll-Hypaque (GE-Healthcare, USA) density gradient centrifugation. After centrifugation, the nucleated cells and deciduous endometrium suspended in the buffy coat were transferred to a new test tube and washed twice with PBS. The cells were suspended in DMEM-F12 medium (Thermo Fisher Scientific, USA) supplemented with 20% FBS (Thermo Fisher Scientific, USA). 5,000 cells were used to improve oocyte quality using a hanging drop culture system.

[0159] C57 / BL6 mice were perfused with PMSG (5 IU / female). Female mice were killed by cervical dislocation 48 hours later. Alcohol was sprayed into the abdomen of the mouse, and an incision was made to expose the abdominal cavity. The ovaries were dissected with a needle and cut into small pieces. Germinal cyst (GV) oocytes were selected and controlled (only medium) or co-cultured with adult stem cells. Before co-culture, 85 IU / ml hyaluronidase was used to remove the cumulus cells around the oocytes in M2 culture medium (Sigma-Aldrich, USA). In order to simulate poor oocyte quality (such as by inhibiting mitochondrial respiratory function), oocytes were treated with 0.05 μM concentration of rotenone for 2 hours. Oocytes were collected after 24 hours of co-culture.

[0160] result

[0161] Scanning electron microscopy confirmed the formation of tunnel nanotubes between menstrual blood cells and oocytes. Fig.11 ). The data also showed that by transferring autologous biomolecules and cellular components from human menstrual blood-derived cells (MBDCs), the maturation rate was improved to be comparable to that of adult stem cells (hASCs) ( Fig.12 ).

[0162] The embodiments and implementations described herein should be understood to be for illustrative purposes only, and the various modifications or changes made according to these embodiments and implementations are self-evident to those skilled in the art, and these modifications or changes should be included in the spirit and scope of the present application. In addition, any element or limitation of any invention disclosed herein or its implementation can be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or its implementation, and it is believed that all these combinations are within the scope of the present invention but do not limit the scope of the present invention.

[0163] References

[0164] Asgharzadeh, S., Mirshokraei, P., Hassanpour, H., Ahmadi, E., & Nazari, H. (2015). The effect of mesenchymal stem cells as co-culture in in vitro nuclear maturation of ovine oocytes. Animal Science Papers and Reports, 33(3).

[0165] Craven, L., et al., Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 2010. 465(7294): p.82-5.

[0166] Engelstad, K., et al., Attitudes toward prevention of mtDNA-related diseases through oocyte mitochondrial replacement therapy. Hum Reprod, 2016. 31(5): p. 1058-65.

[0167] Ling, B., Feng, DQ, Zhou, Y., Gao, T., Wei, HM, & Tian, ​​ZG (2008). Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte. Brazilian Journal of Medical and Biological Research, 41(11), 978-985.

[0168] Rajabi, Z., Yazdekhasti, H., Mugahi, SMHN, Abbasi, M., Kazemnejad, S., Shirazi, A., ... & Zarnani, AH (2018). Mouse preantral follicle growth in 3D co-culture system using human menstrual blood mesenchymal stem cells. Reproductive biology, 18(1), 122-131.

[0169] Wang, ZB, Hao, JX, Meng, TG, Guo, L., Dong, MZ, Fan, LH, ... & Yao, YQ (2017). Transfer of autologous mitochondria from adipose tissue-derived stem cells rescues oocyte quality and infertility in aged mice. Aging (Albany NY), 9(12), 2480.

[0170] Woods, DC, & Tilly, JL (November 2015). Autologous germline mitochondrial energy transfer (AUGMENT) in human assisted reproduction. In Seminars in reproductive medicine (Vol. 33, No. 6, p. 410). NIH Public Access.

Claims

1. A method for generating a mature oocyte from an immature oocyte, the method comprising: One or more immature oocytes are cultured in in vitro maturation (IVM) medium. transfecting one or more tunneling nanotube-forming cells with a vector carrying a microtubule motor protein kinesin family member 5B KIF5B gene, thereby inducing overexpression of KIF5B in the one or more tunneling nanotube-forming cells; as well as co-culturing the one or more immature oocytes with the transfected tunneling nanotube-forming cells for at least one hour under standard culture conditions, such that at least one tunneling nanotube-forming cell forms at least one tunneling nanotube to at least one immature oocyte and transfers biomolecules, cellular components and / or organelles to the at least one immature oocyte through the tunneling nanotube, the tunneling nanotube protruding from the at least one tunneling nanotube-forming cell to the at least one immature oocyte, The at least one tunnel nanotube-forming cell is an adipose stem cell, a bone marrow mesenchymal stem cell, or an adult stem cell derived from a cell population isolated from the menstrual blood of an oocyte donor.

2. The method according to claim 1, wherein: At least one of the tunneling nanotube-forming cells is an autologous cell of a patient undergoing assisted reproductive technology treatment.

3. The method according to claim 1, wherein: The biomolecules and cellular components include one or more nucleic acids, proteins, lipids, carbohydrates, or cellular organelles including mitochondria, or combinations thereof.

4. The method according to claim 1, wherein: The IVM culture medium includes follicle stimulating hormone FSH and serum supplement.

5. The method according to claim 1, wherein: The IVM culture medium also includes an extracellular matrix component.

6. A method for generating a mature oocyte from an immature oocyte, the method comprising: One or more immature oocytes are cultured in IVM medium. culturing one or more tunneling nanotube-forming cells under standard culture conditions; transfecting the one or more tunneling nanotube-forming cells with a vector carrying a microtubule motor protein kinesin family member 5B KIF5B gene; combining the one or more immature oocytes and the one or more transfected tunneling nanotube-forming cells together in a droplet of culture medium, and suspending the drop of the culture medium from an inverted surface for a period of at least one hour such that the one or more immature oocytes and the one or more tunneling nanotube-forming cells are in close proximity in the hanging drop and at least one tunneling nanotube-forming cell forms at least one tunneling nanotube to at least one immature oocyte and transfers biomolecules, cellular components and / or organelles to the at least one immature oocyte through the at least one tunneling nanotube, the tunneling nanotube protruding from the at least one tunneling nanotube-forming cell, The at least one tunnel nanotube-forming cell is an adipose stem cell, a bone marrow mesenchymal stem cell, or an adult stem cell derived from a cell population isolated from the menstrual blood of an oocyte donor.

7. A method for generating a mature oocyte from an immature oocyte, the method comprising: One or more immature oocytes are cultured in IVM medium. culturing one or more tunneling nanotube-forming cells under standard culture conditions; transfecting the one or more tunneling nanotube-forming cells with a vector carrying a microtubule motor protein kinesin family member 5B KIF5B gene; injecting the one or more oocytes and the transfected tunneling nanotube-forming cells into a microfluidic device, The injection microfluidic device comprises: a) at least one tunneling nanotube formation chamber; b) a cell transport network of channels connected to at least one inlet and at least one outlet and to said at least one tunneling nanotube forming chamber, wherein said cell transport network of channels is a reservoir for cells and cell culture medium and the flow rate of said cells and said culture medium is controlled by said device, and c) a constriction comprising one or more microchannels having a diameter different from the diameter of the channels of the cell transport network; and co-culturing the one or more immature oocytes with the transfected one or more tunneling nanotube-forming cells in the microfluidic device for a period of at least one hour, such that at least one tunneling nanotube-forming cell forms at least one tunneling nanotube to at least one immature oocyte and transfers biomolecules, cellular components and / or organelles to the at least one immature oocyte through the tunneling nanotube, the tunneling nanotube protruding from the at least one tunneling nanotube-forming cell to the at least one immature oocyte, The at least one tunnel nanotube-forming cell is an adipose stem cell, a bone marrow mesenchymal stem cell, or an adult stem cell derived from a cell population isolated from the menstrual blood of an oocyte donor.

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