A method for high yield clinical grade wharton's jelly mesenchymal stem cells culturing

Abstract

A method for proliferating Wharton's jelly mesenchymal stem cells comprises the steps of (a) isolating Wharton's jelly tissues comprising viable Wharton's jelly mesenchymal stem cells; (b) partially digesting the isolated tissues by using first culture medium comprising collagenase type I and antibiotic-antimycotic; (c) preparing a primary culture of Wharton's jelly mesenchymal stem cells in suspension culture by using a second culture medium comprising antibiotic-antimycotic and platelet lysate; (d) expanding the primary culture in the suspension culture; (e) obtaining a subculture from the expanded primary culture; and (f) harvesting viable Wharton's jelly mesenchymal stem cells from the subculture.

Description

[0001] A METHOD FOR HIGH YIELD CLINICAL GRADE WHARTON’S JELLY MESENCHYMAL STEM CELLS CULTURING

[0002] Field of Invention

[0003] The invention relates to cell culture, particularly to a method for culturing mesenchymal stem cells derived from Wharton’s jelly of the umbilical cord.

[0004] Background of Invention

[0005] Stem cells are undifferentiated or partially differentiated cells that can differentiate into many types. They have great potential for regenerative medicine, tissue engineering, and disease treatment. There are two main types of stem cells: embryonic stem cells and adult stem cells. Embryonic stem cells are derived from early-stage embryos and can differentiate into any cell in the body. On the other hand, adult stem cells are found in tissues and organs in the body and have a more limited ability to differentiate into different cell types. An example of adult stem cells is mesenchymal stem cells (MSCs) that are able to differentiate into various mesenchymal lineages, including bone cells (osteoblasts), cartilage cells (chondrocytes), muscle cells (myocytes), fat cells (adipocytes), nerve cells (neurocytes), liver cells (hepatocytes), pancreas cells, heart muscle cells (cardiomyocytes), etc.

[0006] MSCs can be derived from adult tissues such as bone marrow and adipose tissue and from extra-embryonic tissues such as the placenta, umbilical cord, and amniotic membrane. The MSCs in the umbilical cord are mainly found in Wharton’s jelly (WJ), a gelatinous tissue within the umbilical cord that surrounds the blood vessels. It was found that Wharton’s jelly contains a much higher concentration of MSCs than cord blood. The Wharton’s jelly MSCs (WJ-MSCs) share many characteristics with adult MSCs, such as the bone marrow MSCs, and they are spared from pro-aging factors. It also has a higher proliferation rate, greater capacity for differentiation, and reduced immune response compared to other MSCs.

[0007] In addition, Wharton's jelly MSCs (WJ MSCs) have gained significant attention in recent years as a potential source of cells for regenerative medicine, particularly for their use in wound healing and tissue regeneration. However, the low concentration of MSCs in all tissues and the insufficiency of separated MSCs for clinical usage pose a significant barrier to the therapeutic application of MSCs. Thus, periodic infusions of MSCs containing up to several hundred million cells are required to obtain the desired therapeutic impact in specific individuals and conditions.

[0008] MSCs can be expanded in vitro using a cell culture plate and flask to achieve the necessary number of cells for an investigation. However, it is crucial to identify an efficient strategy for large-scale expansion to obtain a high number of cells quickly and cost-effectively without compromising their quality.

[0009] Methods for large-scale expansion of stem cells have been developed over the years. A U.S. Pat. No. 10542743 disclosed a method of isolating and proliferating viable MSCs from Wharton’s jelly. The method includes the steps of obtaining umbilical cord tissue comprising viable cells from Wharton’s jelly, isolating the viable cells in a sample collection media, preparing a primary stock culture using the viable cells, proliferating the primary stock culture to obtain sub-cultured stocks, harvesting the viable cells from the sub-cultured stocks and cryo-preserving the viable cells under cryogenic conditions. However, the isolation method disclosed is rather lengthy as it involves incubating the tissue with collagenase overnight and trypsin on the next day, followed by neutralization of the digestion enzymes with a complete medium containing fetal bovine serum (FBS) and removal of undigested samples by centrifugation before preparation of the primary stock culture. The high levels of agitation and aeration can generate high shear stresses, which can damage cells and affect cell growth and productivity. Besides, the use of FBS also poses a risk of contamination with viruses.

[0010] In view of the above, an improved method of culturing MSCs from Wharton’s jelly is highly desirable. The present invention provides a method of culturing Wharton’s jelly MSCs that produces a high yield of cells and exerts less mechanical stress on the cells during isolation. Summary of Invention

[0011] An object of the invention is to provide a method for isolating and proliferating mesenchymal stem cells from the human umbilical cord, particularly Wharton’s jelly mesenchymal stem cells (WJ-MSCs). The method disclosed herein can minimize mechanical stresses exerted on the WJ-MSCs during the dissociation and isolation of the cells from the umbilical cord specimen to prevent cell damage, poor cell growth, and low productivity. Particularly, the method disclosed herein involves partial digestion of the umbilical cord tissues to partially break down the extracellular matrix (ECM) surrounding the WJ-MSCs, thereby reducing the mechanical stress experienced by the cells during the dissociation and isolation process of the cells. Besides, the method disclosed herein is time-saving as partial digestion can be completed within a duration of as short as 30 minutes.

[0012] Another object of the invention is to provide a method for isolating and proliferating WJ- MSCs originating from the umbilical cord of a single donor on a large scale. This is achieved by using a suspension culture instead of a monolayer culture for the expansion of the WJ- MSCs. The method is particularly able to expand WJ-MSCs isolated from a single umbilical cord to up to 1011cells in 3-4 passages with a seeding density of 5000 cells / cm2. More than 300 vials of cryopreserved WJ-MSCs can be obtained from one umbilical cord specimen.

[0013] Yet another object of the invention is to provide a method for isolating and proliferating WJ- MSCs with better preservation of the mesenchymal stem cell phenotypes and improved reproducibility. Through partial digestion of the umbilical cord specimen that gently and slowly dissociates WJ-MSCs from the umbilical cord, the exposure of the WJ-MSCs to stressful conditions is minimized, thereby allowing the WJ-MSCs to maintain their normal behavior and function during cell proliferation. Accordingly, WJ-MSCs with consistent phenotypes of mesenchymal stem cells are obtainable through the method disclosed herein.

[0014] Still, another object of the invention is to provide a method for isolating and proliferating WJ-MSCs that can be performed without technical difficulties. This is achieved by using suspension culture for the expansion of WJ-MSCs, as suspension cultures are relatively simple to set up and maintain. Besides, suspension cultures result in a homogeneous population of cells because the proliferated cells are evenly distributed in the culture medium and can grow and divide without physical interactions with neighboring cells.

[0015] Yet another object of the invention is to provide a method for isolating and proliferating WJ- MSCs that produces a spheroid culture of mesenchymal stem cells (MSCs). Spheroid culture of MSCs enhances the yield of WJ-MSCs by increasing cell proliferation, improving cell survival, and enhancing the expression of crucial MSC surface markers compared to conventional monolayer culture methods.

[0016] At least one of the preceding objects is met, in whole or in part, in which the embodiment of the present invention describes a method for proliferating Wharton’s jelly mesenchymal stem cells. The method comprises the steps of: (a) isolating Wharton’s jelly tissues comprising viable Wharton’s jelly mesenchymal stem cells from an umbilical cord specimen; (b) partially digesting the isolated tissues by incubating the isolated tissues in a first culture medium comprising a basal medium, 0.1% to 1.0% collagenase type I and 1% to 10% antibiotic-antimycotic by weight of the basal medium for 10 minutes to 6 hours; (c) preparing a primary culture (P0) of Wharton’s jelly mesenchymal stem cells by proliferating the partially digested tissues in a suspension culture having a second culture medium comprising the basal medium, 0.1% - 10% antibiotic-antimycotic and 0.1% - 20% platelet lysate by weight of the basal medium; (d) expanding the primary culture in the suspension culture by periodically adding or replacing the second culture medium and periodically splitting the suspension culture; (e) obtaining a subculture from the expanded primary culture; and (f) harvesting viable Wharton’s jelly mesenchymal stem cells from the subculture.

[0017] In a preferred embodiment of the invention, the method further comprises a step of dissociating the cells in the primary culture before the step (e), a step of dissociating the cells in the subculture before the step (f), or both.

[0018] In a preferred embodiment of the invention, the method further comprises a step of cry opreserving the harvested Wharton’s jelly mesenchymal stem cells. In a preferred embodiment of the invention, when the second culture medium is replaced in step (d), the cells in the replaced culture medium are recovered and further expanded in the suspension culture.

[0019] In a preferred embodiment of the invention, the subculture is obtained by seeding the cells from the expanded primary culture in the suspension culture at a density of 4 x 105cells / mL to 8 x 105cells / mL.

[0020] In a preferred embodiment of the invention, the steps of (c) to (e) are performed using a plurality of cell culture flasks having a cell growth surface area of 20 cm2to 80 cm2.

[0021] In a preferred embodiment of the invention, the primary culture and the subculture comprise spheroids.

[0022] In a preferred embodiment of the invention, 80% or more of the harvested cells express at least one surface marker selected from the group consisting of CD44, CD73, CD90 and CD105.

[0023] The present invention further describes a tissue digestion medium for use in a method for proliferating Wharton’s jelly mesenchymal stem cells described in the preceding paragraphs. The tissue digestion medium comprises a basal medium, 0.1% to 1.0% collagenase type I, and l%to 10% antibiotic-antimycotic by weight of the basal medium.

[0024] The present invention describes a culture expansion medium for use in a method for proliferating Wharton’s jelly mesenchymal stem cells described in the preceding paragraphs.

[0025] The culture expansion medium comprises a basal medium, 1% antibiotic -antimycotic, and 5% platelet lysate by weight of the basal medium.

[0026] Still, other objects and advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein the preferred embodiments of the invention are shown and described simply by an illustration of the best mode of carrying out the invention. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious aspects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative and not as restrictive.

[0027] Brief Description of Drawings

[0028] To facilitate an understanding of the invention, the accompanying drawings illustrate the preferred embodiments from an inspection, which, when considered in connection with the following description, would readily understand and appreciate the invention, its construction and operation, and many of its advantages.

[0029] Figure 1 illustrates steps (a) to (c) of the method according to a preferred embodiment of the present invention.

[0030] Figure 2 illustrates steps (d) to (e) of the method according to a preferred embodiment of the present invention.

[0031] Figure 3 shows the morphology of proliferated Wharton’s jelly mesenchymal stem cells (WJ-MSCs) in the Cl, C2, C3A, and C3B flasks of the primary culture (passage 0, P0) obtained in step (d) of the method. Each cell morphology represents different stages of cell and medium transition during the culture.

[0032] Figure 4 shows the morphology of proliferated WJ-MSCs in the secondary subculture (passage 1, P2) obtained in step (e) of the method.

[0033] Figure 5 depicts an increase in the number of proliferated WJ-MSCs obtained by the method according to a preferred embodiment after multiple passages for three samples derived from different umbilical cord specimens, in which (A) shows the number of proliferated cells after each passage for the three samples, and (B) shows the accumulative cell count from three samples through passage 3. Figure 6 shows the flowcytometry histograms for the expression levels of CD 13, CD29, CD44, CD73, CD90, CD105, CD14, CD34, CD45, and HLA-DR in the proliferated WJ-MSCs obtained by the method according to a preferred embodiment of the invention. The numbers in the top left comer indicate the percentage of positive cells.

[0034] Figure 7 shows the trilineage differentiation of the proliferated WJ-MSCs obtained by the method according to a preferred embodiment of the invention. Oil red O, alizarin red, and safranin O staining demonstrate the trilineage differentiation potential of the proliferated WJ-MSCs.

[0035] Detailed Description of Invention

[0036] Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages and those inherent. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention. It is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim. The embodiment described herein is not intended as a limitation on the scope of the invention.

[0037] The terms “proliferating” and "expanding" as used herein refer to increasing the number of cells in the cell population due to cell replication.

[0038] The present invention describes a method for proliferating Wharton’s jelly mesenchymal stem cells from an umbilical cord specimen. The method comprises the steps of (a) isolating Wharton’s jelly tissues from an umbilical cord specimen; (b) partially digesting the isolated tissues by using a first culture medium; (c) preparing a primary culture of Wharton’s jelly mesenchymal stem cells by using a suspension culture having a second culture medium; (d) expanding the primary culture in the suspension culture; (e) obtaining a subculture from the expanded primary culture; and (f) harvesting viable Wharton’s jelly mesenchymal stem cells from the subculture.

[0039] The umbilical cord specimen can be obtained from any mammal having an umbilical cord with Wharton’s jelly, including but not limited to humans, primates, cattle, buffalo, sheep, and goats. In the preferred embodiment of the present invention, a human umbilical cord specimen is used, and viable human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) are isolated and cultured therefrom using the method disclosed herein. The umbilical cord specimen freshly collected from a donor is preferably in a buffer solution mixture . This buffer solution mixture comprises a substance that inhibits bacterial growth (e.g., antibiotics) and a substance that inhibits fungal growth (e.g., antimycotic). Any buffer solution that maintains pH while minimizing osmotic shock in living cells can be used. Examples of buffer solutions suitable for use in the method disclosed herein are phosphate -buffered saline (PBS) and Dulbecco's phosphate-buffered Saline (DPBS). Preferably, Dulbecco’s phosphate-buffered saline is used. Antibiotic-antimycotic (AA) can prevent the growth of bacteria and fungi in the umbilical cord specimen. Preferably, the antibiotic-antimycotic comprises a mixture of penicillin, streptomycin, and amphotericin B.

[0040] According to the preferred embodiment, the method disclosed herein comprises a step of isolating Wharton’s jelly tissues from the umbilical cord specimen (Step (a)). Preferably, the isolated Wharton’s jelly tissues comprise viable Wharton’s jelly mesenchymal stem cells. Prior to step (a), the umbilical cord specimen is removed from the mixture of buffer solution, antibiotic, and antimycotic and cut into multiple segments. Preferably, the umbilical cord specimen is cut into segments of 2 cm to 8 cm, preferably segments of 5 cm. The umbilical cord segments are further disinfected to remove blood contaminants therefrom. Preferably, the cord segments are wiped with an alcohol swab and then rinsed with a buffer or washing solution to minimize damage to the viable cells in the cord segments during disinfecting. The buffer solution can be any buffer solution as long as it does not compromise the workability of the method and the viability of the cells in the cord segments. Examples of buffer solutions suitable for use in the method disclosed herein include PBS and DPBS. Optionally, blood contaminants can be removed from the cord segments by washing the cord segments with double distilled water multiple times and then repeatedly immersing the cord segments in a buffer solution containing antibiotic-antimycotic (AA), penicillin-streptomycin (Pen-Strep) or a mixture thereof.

[0041] Pursuant to the preferred embodiment of the invention, in step (a) of the method disclosed herein, disinfected umbilical cord segments are cut open, and only the Wharton’s jelly tissues containing viable mesenchymal stem cells are harvested therefrom. Preferably, the harvested Wharton’s jelly tissues are further cut or minced into smaller pieces of 1 mm to 5 mm, preferably 2 mm to 3 mm.

[0042] According to the preferred embodiment, the method comprises a step (b) of partially digesting Wharton’s jelly tissues obtained in step (a) through a short digestion enzyme exposure to further isolate and liberate viable mesenchymal stem cells from the extracellular matrix. In the method disclosed herein, partial enzymatic digestion is preferred over complete enzymatic digestion as it creates less mechanical stress to the Wharton’s jelly mesenchymal cells, which can adversely affect cell growth and productivity in the subsequent culturing process. Particularly, the Wharton’s jelly tissues are added into a first culture medium and incubated at 20 °C to 40 °C for 10 minutes to 6 hours. Preferably, the partial enzymatic digestion of Wharton’s jelly tissues is conducted at 37 °C for 2 hours or less. More preferably, partial enzymatic digestion is allowed for 1 hour or less. Most preferably, the Wharton’s jelly tissues are subjected to partial digestion for 30 minutes or less.

[0043] Preferably, the first culture medium used in step (b) is a tissue digestion solution comprising a basal medium added with collagenase type I and antibiotic-antimycotic. Examples of basal medium suitable for preparing the first culture medium include but are not limited to Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM: Fl 2). Preferably, the first culture medium comprises 0.1% to 1.0% collagenase type I and l% to 10% antibiotic- antimycotic by weight of the basal medium. Collagenase type I is the preferred digestion enzyme in the method disclosed herein. It is a relatively gentle digestion enzyme that digests native triple -helical collagen fibrils in connective tissue without agitation. Unlike trypsin, which hydrolyses cell membrane proteins, collagenase type I breaks the peptide bonds of collagen contained within the extracellular matrix, leaving the cell membrane of the digested Wharton’s jelly tissues intact. Accordingly, a higher initial amount of viable Wharton’s jelly mesenchymal stem cells is available for subsequent cell proliferation.

[0044] The method disclosed herein further comprises a step (c) to prepare a primary culture (P0) of Wharton’s jelly mesenchymal stem cells from the partially digested Wharton’s jelly tissues obtained in step (b). The primary culture is particularly obtained by proliferating the dissociated Wharton’s jelly mesenchymal stem cells in the partially digested tissues in suspension culture with a second culture medium. The partially digested Wharton’s jelly tissues may or may not be isolated from the first culture medium prior to the preparation of the primary culture. The second culture medium may be directly added to the mixture of the partially digested tissues, and the first culture medium may be used to form the suspension culture. The presence of collagenase type I in the first culture medium does not impair the proliferation of the viable mesenchymal stem cells in the partially digested tissues in the suspension culture. Subsequently, the mixture of partially digested tissues and the first culture medium may be subjected to centrifugation to obtain a pellet of partially digested Wharton’s jelly tissues that can be resuspended in the second culture medium to form the suspension culture. Besides, the mixture of the partially digested tissues and the first culture medium contains an extracellular matrix of Wharton’s jelly tissues, which facilitates the proliferation of the mesenchymal stem cells.

[0045] Preferably, the second culture medium is a cell culture medium comprising a basal medium, antibiotic-antimycotic, and platelet lysate. More preferably, the second culture medium comprises 0.1% - 10% antibiotic-antimycotic and 0.1% - 20% platelet lysate by weight of the basal medium. Examples of basal medium suitable for preparing the first culture medium include but are not limited to Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM: Fl 2). An antibiotic -antimycotic mixture of penicillin, streptomycin, and amphotericin B is preferred in the second culture medium. Preferably, the second culture medium comprises human platelet lysate (HPL). HPL provides a better mimicry of human biology, thereby boosting the growth and survival of Wharton’s jelly mesenchymal stem cells to form a quality primary culture. Preferably, HPL contains growth factors, such as platelet- derived growth factor (PDGF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF), which enhance cell proliferation and promote cell growth. HPL also contains anti- apoptotic elements, such as interleukin- 1 beta (IL-lbeta), that can help to promote cell survival. HPL can reduce the reliance on exogenous growth factors, as platelet lysate contains a variety of growth factors and cytokines essential for cell growth and proliferation. Besides, HPL poses a lower-risk source of contamination than fetal bovine serum (FBS), which is commonly used in mammalian cell culture media.

[0046] Preferably, in step (c) of the method disclosed herein, the primary culture is obtained by incubating the suspension culture comprising the partially digested tissues and second culture medium at 37 °C in the presence of humidified carbon dioxide until the cell confluency reaches 80% or more. Preferably, the suspension culture is transferred into at least one cell culture flask with a cell growth surface area of 20 cm2to 30 cm2, preferably 25 cm2, before the incubation. The resulting primary culture is labeled as Passage 0 (P0).

[0047] Figure 1 depicts one preferred embodiment of the invention. The umbilical cord specimen is divided into segments. Wharton’s jelly tissues are harvested from the cord segments and further minced to the size of 2 mm2to 3 mm2. The first culture medium is added into a plurality of centrifuge tubes. The minced Wharton’s jelly tissues are added into the centrifuge tubes for partial digestion with collagenase type I. Particularly, the partial digestion is performed in an incubator shaker by spinning the centrifuge tubes at an angle of 45° at a rotation speed of 250 rpm at 37 °C for 30 minutes. After the partial digestion, the supernatant containing the first culture medium is removed from the centrifuge tubes, leaving a pellet containing the partially digested Wharton’s jelly tissues in the centrifuge tubes. The pellet is then resuspended in the second culture medium and transferred into a plurality of cell culture flasks (T25cm) with a cell growth surface area of 25 cm2. This first set of cell culture flasks is labeled as Cl. The suspension culture in the Cl flasks is incubated at 37 °C in 5% humidified carbon dioxide until the confluency of cells reaches 80% or more to obtain a primary culture.

[0048] Pursuant to the preferred embodiment of the invention, the method of the invention comprises a step (d) of expanding the primary culture (P0) obtained in step (c) in suspension culture in the incubator by continuously supplementing new growth nutrients and growth space for the proliferation of Wharton’s jelly mesenchymal stem cells. Preferably, the suspension culture comprises the second culture medium described in the preceding paragraph. The second culture medium comprises growth nutrients that support the proliferation of the cells in the suspension culture. As the stem cell proliferation proceeds, the growth nutrients in the second culture medium diminish and eventually are fully spent.

[0049] In particular, expanding the primary culture involves maintaining the primary culture by periodically adding a fresh second culture medium into the suspension culture or replacing the spent second culture medium in the suspension culture with a fresh second culture medium. It is preferred that after adding a fresh second culture medium to the suspension culture two to three times, the second culture medium in the suspension culture is replaced at least once in order to remove toxic biological products accumulated in the suspension culture. Preferably, when the second culture medium is replaced in step (d), the proliferated mesenchymal stem cells in the replaced or spent culture medium are recovered and further expanded in the suspension culture. More preferably, the culture medium from different cell culture flasks is pooled together during the medium change step, and the proliferated mesenchymal stem cells are recovered from the pooled culture medium.

[0050] In one exemplary embodiment, the spent second culture medium in the suspension culture is replaced with a fresh second culture medium by separating a pallet of proliferated mesenchymal stem cells from the suspension culture via centrifugation and then resuspending the pallet of cells in the fresh second culture medium to form a new suspension culture. The supernatant obtained from centrifugation of the spent culture medium is pooled together. The supernatant is subjected to an additional round of centrifugation to recover the remaining proliferated mesenchymal stem cells. Other methods of separating the proliferated mesenchymal stem cells from the suspension culture can be used as long as the separation methods do not impair the function and performance of the method disclosed herein.

[0051] Furthermore, the step of expanding the primary culture comprises periodically splitting or dividing the suspension culture when the suspension culture reaches a cell confluency of 80% or more. The suspension culture of the primary culture may be split and transferred into multiple cell culture flasks. A fresh second culture medium is added directly into the flask to maintain and further expand the primary culture. Preferably, the suspension culture is distributed fairly evenly to each cell culture flask. Alternatively, the suspension culture is subjected to centrifugation to separate a pallet of proliferated mesenchymal stem cells from therefrom. A new suspension culture is obtained by resuspending the pallet of cells in a fresh second culture medium. The new suspension culture is then further distributed to multiple cell culture flasks. Additional fresh second culture medium may be added to the flasks. Preferably, the cell culture flasks have a cell growth surface area of 20 cm2to 30 cm2, preferably 25 cm2. It is preferred that the primary culture is expanded to a total volume of 3 mb to 7 mb with cell confluency of 80% or more.

[0052] In the preferred embodiment of the invention, the method comprises step (e) of obtaining at least one subculture from the expanded primary culture obtained in step (d). The expanded primary culture is subcultured by harvesting the proliferated mesenchymal stem cells therefrom, reseeding the harvested cells in a fresh cell culture medium to form a suspension culture, and proliferating the reseeded cells in the suspension culture until the cell reaches a confluency of 80% or more. Preferably, the subculture is obtained by reseeding the harvested cells in the cell culture medium at a density of 4 x 105cells / mL to 8 x 105cells / mL. The reseeded cells are proliferated in the suspension culture in order to obtain the subculture. The harvested cells are preferred to be reseeded into a plurality of cell culture flasks with a cell growth surface areaof 70 cm2to 80 cm2or, more preferably, 75 cm2. The cell culture medium used to form the suspension culture for the subculture can be any cell culture medium that supports mesenchymal stem cell proliferation. Preferably, the second culture medium described in the preceding description is used to prepare the subculture(s).

[0053] The expanded primary culture is subcultured once into a secondary culture or Passage 1 (Pl) in the invention's preferred embodiment. Optionally, the secondary culture (Pl) is further subcultured into a tertiary culture or Passage 2 (P2), quaternary culture or Passage 3 (P3), quinary culture or Passage 4 (P4), and so on. The method of the present invention is advantageous in that sufficient proliferated mesenchymal stem cells are obtainable after the first and second subcultures. In one embodiment of the invention, when a seeding density of 1.8 x 106to 2 x 106cells is used, 5.5 x 108to 6 x 108cells can be obtained in the first subculture (Pl), and 7.5 x 109to 8 x 109cells can be obtained in the second subculture (P2). The method disclosed herein promotes spheroids formation in the suspension culture. The proliferated Wharton’s jelly mesenchymal stem cells aggregate and self-assemble into three- dimensional (3D) sphere-like formations that freely float in the suspension culture. The formation of 3D cell clusters improves cell proliferation and cell survival. Furthermore, the spheroid culture of mesenchymal stem cells enhances the expression of crucial mesenchymal stem cell markers, thereby increasing the number of proliferated mesenchymal stem cells. Preferably, both the primary culture and the subculture(s) obtained from steps (d) and (e) of the method comprise spheroids.

[0054] The method disclosed herein further comprises a step of dissociating the cells in the expanded primary culture obtained in step (d) before the step (e) and / or a step of dissociating the cells in the expanded subculture(s) obtained in step (e) before the step (f). Preferably, the expanded cultures are subjected to a treatment with trypsin to dissociate the cell aggregates in the suspension culture. Optionally, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) is used in conjunction with trypsin to facilitate trypsin in dissociating cell aggregates. Following the trypsinization treatment, trypsin inhibitors are added to the suspension culture to protect the dissociated cells against further protein degradation by trypsin.

[0055] Figure 2 illustrates one preferred embodiment of the invention. After the primary culture (P0) in the Cl flasks reaches a cell confluency of 80% or more, the fresh second culture medium is added into the flasks on the following 4 days, i.e., days 2 to 5, in order to maintain the primary culture. Optionally, the second culture medium added into the Cl flasks on days 3 to 5 contains no platelet lysate. Subsequently, the suspension culture in the Cl flasks is split by transferring about half of the suspension culture into a second set of T25 flasks (C2 flasks). A few days following the division, the spent culture medium in the Cl and C2 flasks is replaced with a fresh second culture medium, and the primary culture in C 1 and C2 flasks are further proliferated for more than 15 days, preferably 16 to 30 days, or more preferably 16 to 21 days. Preferably, the spent culture medium is removed from the suspension culture by centrifugation, and the pellets of cells are resuspended in a fresh second culture medium. The replaced culture medium collected from the Cl and C2 flasks, i.e., the supernatant of the suspension culture, is pooled together and subjected to centrifugation. The recovered pellet of mesenchymal stem cells is resuspended in a fresh second culture medium in a third set of T25 flasks (C3A flasks) for further proliferation. Optionally, the suspension culture in the Cl, C2, and C3A flasks is subjected to another cycle of centrifugation after a few days of cell proliferation. The supernatant is pooled together, and proliferated cells recovered from the pooled supernatant are further proliferated in a fourth set of T25 flasks (C3B flasks). The suspension culture in the Cl, C2, C3A, and C3B flasks is subjected to trypsinization when cell confluency of 80% or more is reached. The proliferated cells are harvested from the trypsinized suspension culture and reseeded in a plurality of cell culture flasks (T75 flasks) with a cell growth surface area of 70 cm2to 80 cm2, preferably 75 cm2. A Passage 1 (Pl) subculture is obtained by proliferating the reseeded cells in the T75 flasks until the cells reach a confluency of 80% or more.

[0056] After the subculturing in step (e), the method comprises a step (f) of harvesting viable Wharton’s jelly mesenchymal stem cells from the subculture. Particularly, the subculture is subjected to a trypsinization treatment as described in the preceding description, followed by a centrifugation process to separate the viable proliferated mesenchymal stem cells from the suspension culture. The resulting pallet of viable cells is stored appropriately to ensure the cells' viability. Preferably, the method disclosed herein further comprises a step of cryopreserving the harvested viable mesenchymal stem cells. One cryovial is preferred to contain 1.8 x 106to 2.4 x 106cells per mb of the cryopreservation medium.

[0057] According to the preferred embodiment of the invention, Wharton’s jelly mesenchymal stem cells obtained by the present invention's method express phenotypes unique to mesenchymal stem cells. More than 90% of the viable cells harvested in step (f) express surface markers selected from the group consisting of CD44, CD73, CD90 and CD105. Besides, less than 5% of the harvested cells express negative surface markers selected from the group consisting of CD45, CD34, CDl lb, CD19 and HLA-DR. In more particular, the Wharton’s jelly mesenchymal stem cells proliferated by using the method disclosed herein express phenotypes including, but not limited to, CD44(+), CD73(+), CD90(+), CD105(+), CD45(-), CD34(-), CDl lb(-), CD19(-), HLA-DR(-). In addition, Wharton’s jelly mesenchymal stem cells obtained from the method of the invention exhibit adipogenic, chondrogenic, and osteogenic differentiation. According to the preceding description, the present invention also relates to a tissue digestion medium for use in proliferating Wharton’s jelly mesenchymal stem cells. The tissue digestion medium disclosed herein comprises a basal medium, 0.1% to 1.0% collagenase type I, and 1 % to 10% antibiotic-antimycotic by weight of the basal medium . Examples of basal medium suitable for preparation include but are not limited to, Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM: F12).

[0058] According to the preceding description, a culture expansion medium is used to proliferate Wharton’s jelly mesenchymal stem cells. The culture expansion medium disclosed herein comprises a basal medium, 1% antibiotic -antimycotic, and 5% platelet lysate by weight of the basal medium. Examples of basal medium suitable for preparing the culture expansion medium include but are not limited to Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM: F12).

[0059] Examples

[0060] Below are examples to illustrate different aspects and embodiments of the invention. The examples are not intended to limit the disclosed invention, which is limited only by the claims.

[0061] Example 1 : Sample procurement

[0062] Umbilical cord samples (approximately 35 cm to 40 cm) are collected from healthy donors. Collection kits with sample tissues are maintained on cold packs in the sample collection box and transported immediately to the cGMP facility at HOPE LIFESCIENCE SDN. BHD. Quality Control personnel shall receive the sample, perform a visual inspection for a proper bottle seal, and assess any possible physical damages to the sample packaging. The donor blood was sent to Gribbles for RPR (VDRL), HIV I & II antigen / antibody (Ag / Ab) combo screening, Hepatitis B Surface Antigen Screening (HBsAg), Hepatitis C Antibody Screening (Anti-HCV), Hepatitis B Surface Antibody (Anti-HBs), Cytomegalovirus, CMV (IgG & IgM), ABO group and Rh type.

[0063] Example 2: Cleaning and isolation process The umbilical cord is cut 5 cm long in each process and put on the petri dish. The umbilical cord is cleaned with an alcohol swab and rinsed with 20 mb Dulbecco’s phosphate buffered saline (DPBS) 2 times. The cord is transferred to a new dish each time. The cord is cut and opened to take only Wharton’s jelly part. The Wharton’s jelly is cut into smaller pieces 2-3 mm so that a 3 mL Pasteur pipette can take them up. A Pasteur pipette is used to transfer the cut pieces into a 50 mL centrifuge tube. Every tube contains 5 mL in volume. A digest solution of collagenase type I 0.6% in F12: DMEM + 6% AA is prepared. Each 50 mL centrifuge tube that contains Wharton’s jelly is added with 1 mL of the digest solution. The centrifuge tubes are put in an incubator shaker at 37 °C for 30 minutes. After 30 minutes, 6 mL growth medium (F12:DMEM+1% AA+5% Elitegrow or any growth media) is added into each tube, and then 4 mL of the content therein is transferred into three T25cm flasks separately. The T25cm flasks have a cell growth surface area of 25 cm2. The flasks are incubated in a CO2 incubator and labeled as P0 (Cl). (Figure 1)

[0064] Example 3: Expansion, harvest, and cryopreservation process

[0065] The Cl flasks are observed under an inverted microscope daily to check for microcontamination. The flask is discarded immediately if it is contaminated. After 2 days, 2mL F12: DMEM+1% AA+5% Elitegrow is added into the flasks' suspension culture (passage 0, P0), and the suspension culture is incubated again. On the 3rd to 5th days, the suspension culture in the flasks is transferred into new T25 flasks. 5 ml F12: DMEM+1% AA+5% Elitegrow is added to the Cl flask and mixed. Half of the suspension culture in the Cl flasks is transferred into C2 flasks, whereby each Cl and C2 flask contains 5 ml of the suspension culture. The P0 suspension culture in the flasks is incubated. Subsequently, the medium in the suspension culture is changed every 3 days till the suspension culture reaches > 80% confluence. Incubation of the P0 suspension culture in the Cl and C2 flasks is prolonged until 21 days. When changing the medium, the spent medium from the Cl and C2 flasks is pooled into a C3A and C3B flask. The spent medium is centrifuged at 2,246 g for 5 minutes to obtain a pellet of cells, which is then resuspended in F12: DMEM+1% AA+5% Elitegrow. The cells from the pellet are cultured inside the incubator at 37°C in 5% humidified carbon dioxide until the confluency of the cells reaches > 80%. The cells in the Cl, C2, C3A, and C3B flasks are trypsinized using Accutase. Particularly, the spent medium in the flasks is removed, Accutase is transferred into the flasks, and the mixture is incubated for 5 minutes in biological safety cabinets. If more than 50% of cells have detached from the flask, the flask is tapped to detach more cells. The cells are transferred into a 50 mL centrifuge tube together with Accutase. The flasks are rinsed with spent medium, and the spent medium is also transferred into the centrifuge tube. Cell count and viability are performed. The centrifuge tube is spun at 2246 g for 5 minutes. The supernatant is discarded, and F12: DMEM+1% AA+5% Elitegrow is added into the centrifuge tube to resuspend the pellet of cells. The resulting suspension expands the primary culture to passage 1 (Pl) in T75 flasks with a cell growth surface area of 75 cm2. A seeding density of 4 x 105 / flask to 8 x 105 / flask is used, and the medium is changed every 2 or 3 days until the Pl culture reaches> 80% confluence. Upon confluence at Passage 1, which is > 80% confluency, the cells are trypsinized and then cryopreserved with NutriFreeze D10. One cryovial contains 1.8 x 106cells to 2.4 x 106cells per every 1 ml of NutriFreeze D10. The cryovial s are placed inside CoolCells and stored in a -800 °C deep freezer for at least 4 hours before being transferred to the cryo tank. (Figure 2)

[0066] Example 4: Morphology of the Wharton’s jelly mesenchymal stem cells during the expansion of primary culture (P0) and passage 1 subculture (Pl)

[0067] The morphology of the proliferated Wharton’s jelly mesenchymal stem cells in Cl, C2, C3A, and C4A flasks are observed. Figure 3 shows the morphology of the cells at different stages of cells and medium transition during the expansion of primary culture (P0).

[0068] The expansion in suspension culture, which consists of collecting the spent medium from each cycle of media transition throughout the culture phase and re-culturing it in a new vessel for the proliferation of more cells, is a key technique utilized during the expansion of primary culture in the present invention. This approach is intended to gather floating cells throughout the culture phase. It is shown in Figure 3 that when the mixture of cells and partially digested tissue was cultivated, more cells were observed in suspension, unable to adhere to the culture plate (Cl and C2 flasks). The phenomena occurred due to the undigested tissue covering the surface of the culture plate, avoiding the adhered floating cells. A few cycles of supernatant were collected, centrifuged, and transferred to a new vessel of a culture plate during the expansion phase. It can be seen in Figure 3 that the cells in the C3A flask begin to aggregate into a spheroid form. This process of pooling, centrifuging, and transferring the supernatant is continued until fewer or no cells are visible under a microscope in each culture vessel utilized during the expansion activity.

[0069] The morphology of proliferated cells in the Passage 1 (Pl) subculture was obtained using seeding densities of 5000 cells / cm2. Figure 4 shows that the cells exhibit rapid growth after subculturing in passage 1. Cells reproduce astonishingly rapidly within a day in culture.

[0070] Example 5: Cell counts of proliferated Wharton’s jelly mesenchymal stem cells at Passage 0 to 3

[0071] Three different umbilical cord specimens (MSC001, MSC002, and MSC003) are used to prepare three primary cultures (P0), which are later subcultured to Passage 3. Cell count is performed after the primary culture (P0), subculture Passage 1 (Pl), subculture Passage 2 (P2), and subculture Passage 3 (P3) are obtained. Figures 5(A) and 5(B) show that the number of proliferated Wharton’s jelly mesenchymal stem cells in the primary culture increases after each subculturing.

[0072] The present invention has established the superiority of half digestion and expansion suspension of culture method in promoting the in vitro proliferation of WJ-MSCs and was able to expand WJ-MSCs from a single cord to -1011cells in 3-4 passages at a seeding density of 5000 cells / cm2(Figure 5). The presented method (half-digestion) focuses on the delayed release from WJ-MSCs of the tissue extracellular matrix (ECM) after a short exposure to enzymatic digestion (Figure 1). The WJ-MSCs transition from the ECM into a culture vessel was monitored regularly during expansion. It is essential because this transition can significantly impact the cells' properties and functionality. When MSCs are released in vitro, they are exposed to a very different environment than their natural niche within the tissue matrix. This can lead to cell behavior and phenotype changes, affecting their ability to differentiate and function properly. Therefore, monitoring the cells during the transition from tissue to culture is essential to maintain their sternness and functional properties. Monitoring the transition from tissue to culture can help maintain the cells’ purity and identity. Besides, less damage is done to the cells (short digestion period), and they are prepared to become more viable after exposure to a new environment. The transition from the microenvironment of tissue to single cells is the key step to expanding a large number of stem cells in the shortest duration).

[0073] Using a small-scale culture system, it is demonstrated that 5000 cells / cm2seeding densities increase cell proliferation and yield from passage 1 until 3 (Figures 4 & 5). In addition, it was discovered that supplementing the culture media with platelet lysate increased the proliferative ability of WJ-MSCs. The use of platelet lysate in therapeutic angiogenesis is safe and well -tolerated in human trials. As MSCs lose their stem cell properties during in vitro culture, engaging them early in cell and gene therapy is preferable. Therefore, implementing a seeding density of 5000 cells / cm2and incorporating platelet lysate in the culture medium, F12: DMEM, the WJ-MSCs are expanded to a clinical dose of ~108cells in approximately 21 days or passage 1.

[0074] Example 6: Characterization of proliferated Wharton’s jelly mesenchymal stem cells

[0075] The characteristics of proliferated Wharton’s jelly mesenchymal stem cells obtained using the method described in Examples 1 and 2 are determined. A comprehensive assessment of cell surface marker expression using flow cytometry is performed. Figure 6 indicated that upscaled WJ-MSCs were positive for representative MSC surface markers, including CD 44, CD 73, CD 90, and CD 105. In human bone marrow mesenchymal stem cells (BM-MSCs), some studies have concluded that adding platelet lysate led to the overexpression of HLA- DR and the downregulation of CD44 expression. However, the platelet lysate in the culture expansion medium used in Example 2 did not influence the expression levels of CD44 or HLA-DR in the proliferated WJ-MSCs, as indicated by the fact that upscaled WJ -MSCs were significantly positive for CD44 but negative for HLA-DR.

[0076] In addition, it was discovered that the expanded WJ-MSCs preserved their multilineage potential and could be encouraged to undergo adipogenic, osteogenic, and chondrogenic differentiation, as determined by karyotype analysis, as shown in Figure 7.

Claims

Claims1. A method for proliferating Wharton’s jelly mesenchymal stem cells, comprising the steps of:(a) isolating Wharton’s jelly tissues comprising viable Wharton’s jelly mesenchymal stem cells from an umbilical cord specimen;(b) partially digesting the isolated tissues by incubating the isolated tissues in a first culture medium comprising a basal medium, 0.1% to 1.0% collagenase type I, and 1% to 10% antibiotic-antimycotic by weight of the basal medium for 10 minutes to 6 hours;(c) preparing a primary culture (P0) of Wharton’s jelly mesenchymal stem cells by proliferating the partially digested tissues in a suspension culture having a second culture medium comprising the basal medium, 0. 1% - 10% antibiotic-antimycotic and 0.1% - 20% platelet lysate by weight of the basal medium;(d) expanding the primary culture in the suspension culture by periodically adding or replacing the second culture medium and periodically splitting the suspension culture;(e) obtaining a subculture from the expanded primary culture; and(f) harvesting viable Wharton’s jelly mesenchymal stem cells from the subculture.

2. The method according to claim 1 further comprising a step of dissociating the cells in the primary culture before the step (e), a step of dissociating the cells in the subculture before the step (f), or both.

3. The method according to claim 1 further comprising a step of cryopreserving the harvested Wharton’s jelly mesenchymal stem cells.

4. The method according to claim 1, wherein when the second culture medium is replaced in step (d), the cells in the replaced culture medium are recovered and further expanded in the suspension culture.

5. The method according to claim 1, wherein the subculture is obtained by seeding the cells from the expanded primary culture in the suspension culture at a density of 4 x 105cells / mL to 8 x 105cells / mL.

6. The method according to claim 1, wherein the steps of (c) to (e) are performed using a plurality of cell culture flasks having a cell growth surface area of 20 cm2to 80 cm2.

7. The method according to claim 1, wherein the primary culture and the subculture comprise spheroids.

8. The method according to claim 1 , wherein 80% or more of the harvested cells express at least one surface marker selected from the group consisting of CD44, CD73, CD90 and CD105.

9. A tissue digestion medium for use in a method for proliferating Wharton’s jelly mesenchymal stem cells according to any one of claims 1 to 8, comprising a basal medium, 0. 1% to 1.0% collagenase type I, and 1% to 10% antibiotic-antimycotic by weight of the basal medium.

10. A culture expansion medium for use in a method for proliferating Wharton’s jelly mesenchymal stem cells according to any one of claims 1 to 8, comprising a basal medium, 0.1% - 10% antibiotic-antimycotic and 0.1% - 20% platelet lysate by weight of the basal medium.

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