Placenta-derived potential cells and preparing method thereof

Abstract

A method for culturing placental potential cell is provided, comprising steps of: (1) obtaining placental cells and/or tissue under aseptic condition; (2) inoculating the placental cells and/or the tissue in a culture medium for culturing, adding cell growth regulators to the culture medium, in such a manner that the placental potential cells grows to make the placental cells and/or the tissue into a proliferative state; (3) culturing the placental potential cells to make the placental potential cells proliferate continuously into cells with characteristics of stem cells. The present invention not only finds the source of human tissues, organs and the continuation of their function, i.e., regenerative potential cells; but also finds a medical and health longevity method, but also finds out the life materials to maintain and support the potential cells, so as to replace drugs with the living material.

Claims

1. A method for culturing placental potential cell, comprising steps of: (1) obtaining placental cells and/or tissue under aseptic condition; (2) seeding the placental cells and/or the tissue in a culture medium for culturing, adding cell growth regulators to the culture medium, in such a manner that the placental potential cells grow to make the placental cells and/or the tissue into a proliferative state; (3) culturing the placental potential cells to make the placental potential cells proliferate continuously into cells with characteristics of stem cells.

2. The method, as recited in claim 1, further comprises a step of: under in-vitro induction conditions, forming various types of tissues by cells which are proliferated.

3. The method, as recited in claim 1, wherein the inoculating the placental cells and/or the tissue in the culture medium for culturing of the step (2) comprises steps of: (2-A) washing the placental cells and/or the tissue with phosphate buffer (PBS) containing at least two types of antibiotics comprising penicillin and streptomycin for at least 2 times, 30 seconds-3 minutes for each time; (2-B) washing the placental cells and/or the tissue to a complete medium containing at least two types of antibiotics comprising the penicillin and the streptomycin for at least 2 times, 30 seconds-3 minutes for each time; (2-C) cutting the placental cells and/or the tissue into explants with a size at a range of 1-5 mm.sup.3; (2-D) placing the organ type explants into a center of the culture microplates of the culture plate in 1-5 mm interval, slightly pressing each of the organ type explants to make each of the organ type explants tightly cling to a surface of the culture plate; (2-E) along each periphery of each of the culture microplates, adding 0.1-1.0 ml the complete medium to each of the culture microplates, preventing the complete medium from contacting with the organ type explants; (2-F) sending the culture plate into an incubator at 37° C. with 1-10% CO.sub.2 to pre-culture for 0.5-2 hours; and (2-G) gently adding the complete medium to each of the microplates, preventing the explants from floating, and adding the cell growth regulator.

4. The method, as recited in claim 1, wherein the inoculating the placental cells and/or the tissue in the culture medium for culturing of the step (2) comprises steps of: (2-a) in order to separate the placental tissue into individual cells, firstly putting the placental tissue into 4° C. pre-cooled phosphate buffered saline (PBS) containing at least two types of antibiotics comprising penicillin and streptomycin, washing for at least 3 times, 30 seconds to 3 minutes for each time; then cutting the placental tissue into minimal organ type explants with a size at a range of 1-5 mm.sup.3, washing for two times with cooled PBS containing at least two types of antibiotics; then sending the placental tissue into 0.1-0.5% neutral protease solution or 0.5-2% collagenase solution which is prepared by sterile PBS for digestion, wherein conditions are 36.5-37° C. constant temperature oscillation for 0.5-3 hours; (2-b) gently mixing the explants by pipetting with a sampler or a straw; or pouring the explants digested with 0.1-0.5% dispase or 0.5-2% collagenase solution (in sterile PBS) onto a stainless steel sieve, grinding the explants with a syringe until the explants are completely discrete and become single cells; keeping mixture containing the single cells and digestive enzyme which is known to skills in the art for 2-8 minutes, discarding precipitated chunks and non-digestible connective tissue; transferring supernatant containing plenty of the single cells and the digestive enzyme to another centrifugal tube; filtering the supernatant once with a stainless steel filter, so as to obtain a mixture of the digestive enzyme and the single cell; (2-c) centrifuging for 3-20 minutes with 1000-3000 r/min at 4° C., discarding supernatant containing the digestive enzyme, spinning the single cells by a vortex with PBS which is pre-cooled at 0.5-6° C.; then adding PBS pre-cooled at 0.5-6° C.; and mixing well; (2-d) centrifuging for 3-20 minutes at 0.5-6° C. with a rate 1000-3000 r/min; discarding supernatant, adding cool PBS, oscillating with a vortex to spin the single cells; then adding PBS pre-cooled at 0.5-6° C.; mixing well; and counting the single cells; (2-e) centrifuging for 3-20 minutes at 0.5-6° C. with a rate 1000-3000 r/min; discarding supernatant, adding alpha MEM culture solution containing 10-20% fetal bovine serum; oscillating by vortex to spin up the single cells, then adding the alpha MEM culture solution containing 10-20% fetal bovine serum to regulate the cell concentration at 1×10.sup.5/ml, mixing well to obtain a quantitative cell suspension; (2-f) adding the cell suspension to a plastic porous plate which is a 96-well plate, 24-well plate, 12-well plate or a 6-well plate; wherein adding 200 μl to each well of the 96-well plate; adding 1 ml to each well of the 24-well plate; adding 2 mL to each well of the 12-well plate and adding 4 mL to each well of the 6-well plate; (2-g) sending the plastic porous plate to a cell incubator containing 5% CO.sub.2 at 36.5-37° C. for 12-48 hours until all the cells are adherent.

5. The method, as recited in claim 1, wherein a source of the placenta comprises: a spontaneous abortion human placenta, a mice placenta, a hamster placenta, a pig placenta or a cow placenta.

6. The method, as recited in claim 1, wherein components of the medium comprise: 1) amino acids selected from one or more member from the group consisting of: arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine; 2)vitamins selected from one or more member from the group consisting of: biotin, choline, folic acid, nicotine, pantothenic acid, vitamin B6, vitamin B1 and riboflavin; 3)salt selected from one or more member from the group consisting of: NaCl, KCl, NaHPO.sub.4, NaHCO.sub.3, CaCl.sub.2 and MgCl.sub.2; 4)salt selected from one or more member from the group consisting of: insulin and transferrin; 5) 1 glucose, penicillin and streptomycin.

7. The method, as recited in claim 1, wherein the cell growth regulator is selected from the group consisting of: resveratrol, amino acids, fetal bovine serum and human serum.

8. The method, as recited in claim 1, wherein a content of the resveratrol in the medium is 10-60 ng/mL.

9. A placental potential cell prepared according to the method of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1 is a sketch view showing manufacturing process of potential cells, wherein the potential cells are potential cells with proliferating ability invented by the present invention.

[0109] FIG. 2 is a first microscopic diagram after cultured by the experimental group, wherein the cells are capable of performing sustained proliferation and division and differentiating into various tissues.

[0110] FIG. 3 is a second microscopic diagram after cultured by the experimental group, wherein the cells are capable of performing sustained proliferation and division and differentiating into various tissues.

[0111] FIG. 4 is a sketch view of two pairs of adherent placental potential cells by adding fat inducing agent, wherein the two pairs of adherent placental potential cells are fixed after culturing for 21 days, and then stained with oil red, wherein oil red is dissolved in lipids and insoluble in water, and is commonly used for lipid staining; wherein lipid droplets are found in the differentiated cells in the FIG. 4. (Magnified multiple 10×)

[0112] FIG. 5 is a microscopic image of the osteogenic tissue of the experimental group in the second preferred embodiment; wherein after an identical batch of cells adhered, adding osteogenic inducer, culturing for 21 days and fixing, staining with alizarin red, wherein alizarin red is capable of chelating with calcium ions to form a complex. (Magnified multiple 10×).

[0113] FIG. 6 is an image of stained cartilage tissue in the preferred embodiment 2, wherein the cartilage tissue is formed by adding inducer to cells, culturing for 21 days and staining.

[0114] FIG. 7A-I is a sketch view of cells cultured for 30 days according to the preferred embodiment 3, wherein cell surface markers are determined by flow cytometry. Referring to FIG. 7A-I, the cells in experimental groups express CD90, CD73, CD105, and fail to express CD31, CD45, CD14, CD34, CD9 and HLA-DR.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0115] In order to facilitate the understanding of those skilled in the art, the technical solution adopted by the present invention is to obtain human in situ organ tissue cells; culture the tissue cells in vitro, promote placental potential cell cloning to proliferate functional tissue; extract proteins wherein the extracted proteins is capable of promoting cell growth and tissue repair.

Embodiment 1

Culturing Human Placental Cells in Vitro

[0116] Human placenta is obtained and then performing steps as follows:

[0117] continuously washing the placental tissue with 4° C. pre-cooled phosphate buffer (PBS) containing penicillin and streptomycin for at least 3 times;

[0118] cutting the big block of placental tissue into minimal organ type explants with a size at 2 mm×2 mm×2 mm; and then washing with the 4° C. pre-cooled phosphate buffer (PBS) containing penicillin and streptomycin for at least 2 times;

[0119] sending the placental tissue into 0.25% neutral protease solution or 1% collagenase solution which is prepared by a bacterial PBS for digestion, wherein conditions are 37° C. constant temperature oscillation for 1 hours; and then performing steps as follows:

[0120] repeatedly blowing and beating the minimal organ type explants with a sampler or a straw; or pouring the minimal organ type explants and the neutral protease solution or collagenase solution which is prepared by a bacterial PBS for digestion onto a stainless steel screen, grinding the organ type explants with a syringe until the organ type explants are completely discrete and become single cells;

[0121] standing mixture containing the single cells and digestive enzyme which is known to skills in the art, such as such as neutral protease and collagenase, for 5 minutes, discarding precipitated chunks and non-digestible connective tissue; transferring supernatant containing plenty of the single cells and the digestive enzyme to another centrifugal tube; filtering the supernatant once with a stainless steel filter, so as to obtain a digestive mixture;

[0122] centrifuging for 5 minutes with a rate 1500 r/min at 4° C., discarding supernatant containing the digestive enzyme, spinning the single cells by a vortex with cooled PBS; then adding cooled PBS; and mixing well;

[0123] centrifuging for 5 minutes at 4° C. with a rate 1500 r/min; discarding supernatant, adding cool PBS, oscillating with a vortex to spin the single cells; then adding cooled PBS; mixing well; and counting the single cells;

[0124] centrifuging for 5 minutes at 4° C. with a rate 1500 r/min; discarding supernatant, adding alpha MEM culture solution containing 15% fetal bovine serum; oscillating by vortex to spin up the single cells, then adding the alpha MEM culture solution containing 15% fetal bovine serum to regulate the cell concentration at 1×10.sup.5/ml, mixing well to obtain a quantitative cell suspension;

[0125] adding the cell suspension to a plastic porous plate which is a 96-well plate, 24-well plate, 12-well plate or a 6-well plate; wherein adding 2000 to each well of the 96-well plate; adding 1 ml to each well of the 24-well plate; adding 2 mL to each well of the 12-well plate and adding 4 mL to each well of the 6-well plate;

[0126] sending the plastic porous plate to a cell incubator containing 5% CO.sub.2 at 37° C. for 24 hours until all the cells are adherent.

[0127] After culturing for 24 hours, the adherent cells are observed growing normally under microscope. The culture wells were divided into two groups. One group was the experimental group and the other group was the control group. In the experimental group, alpha MEM medium and resveratrol containing 15% fetal bovine serum are added. The selection of cell culture media is well known to those skilled in the art. The control group did not include the substance of the present invention. The cell culture regulator was added in an amount of 10 ng-60 ng/ml in the medium.

[0128] According to the procedures required, replace the culture medium. A liquid replacement method is removing half of the old culture medium, and adding the same amount of fresh alpha MEM culture medium with 15% fetal bovine serum. Then the culture solution is changed in each 3 days, and combination of regular observation and observation at any time are adopted.

[0129] After 25 days of culture, as shown in FIG. 1, human placental cells are in a single form in the culture medium, at this moment the cells survive in the culture medium, and the life is active, and some cells are in split phase. Some cells in these cells have the ability of continuously proliferating, we call placental potential cells; some cells do not have the ability of continuously proliferating.

[0130] Referring to FIG. 2, it can be seen that the larger cells that are dividing are cells that are replicating the tissues and organs of the placental potential cells; the smaller undifferentiated cells are the original non-proliferating and newly produced placental potential cells; these cells proliferate continuously in culture medium in the role of special biological substances, and perform stem cell proliferation. After the determination of the function, the cells can be used as an in vitro normal cell experimental model.

[0131] Referring to FIG. 3, it can be seen that the cells that are continuously proliferated begin to link into tissue in the culture medium, and the cells that have been linked into the tissue changes their shapes of cells from circular form to forms of the tissue cells, and part of the cells continue to proliferate. The tissue formed can be used as an in vitro tissue experimental model.

[0132] In the study of tissue cells used for tissue organs culture in vitro, the inventors have studied the source cells with proliferative ability which are culture at identical time and found that some cells began to divide into a single tissue cell, and soon became a terminal cell, no new tissues are formed; and the other part of the cells then continue to split and proliferate, and the proliferated cells continuously form tissues, several different forms of tissues together combines into a large tissue.

Embodiment 2

[0133] Formation of Adipose Tissue, Induction and Differentiation of Bone and Cartilage

[0134] Referring to FIG. 3, continuously culture placental cells in the experimental group, continuously add three inducers of bone, cartilage and fat, two weeks later, adipose tissue, bone and cartilage are formed. See FIG. 4-5, respectively using different staining method, bone, cartilage, adipose tissue are formed under the microscope.

Embodiment 3

[0135] Identification of Cell Surface Markers

[0136] Referring to FIG. 7, the control group and the experimental group were set up in the same manner as in Example 1, and the substance of the present invention is not added to the control group. After 30 days of culture, cell surface markers were determined by flow cytometry. Referring to FIGS. 7A-7I, the experimental results are shown as below.

[0137] FIG. 7A shows CD90>95%, FIG. 7B shows CD34<2%; FIG. 7C shows CD14<2%, FIG. 7D shows CD45<2%, FIG. 7E shows CD19<2%, FIG. 7F shows CD105>95%, FIG. 7G shows CD31<2%, FIG. 7H shows CD73>95%, FIG. 7I shows HLA-DR<2%. The experimental group expresses CD90, CD73 and CD105 and CD45, CD14 and HLA-DR are not expressed. The control group do not express any of them.

Embodiment 4

[0138] The origin of the placenta was replaced by a mouse, a hamster, a pig, or other animal, Embodiments 1-3 are repeated and the results are similar to those of Examples 1-3.

[0139] According to another preferred embodiment of the present invention, the components of the medium comprise:

[0140] 1) 80% by mass fraction of amino acids selected from one or more member from the group consisting of: arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine;

[0141] 2) 4% by mass fraction of vitamins selected from one or more member from the group consisting of: biotin, choline, folic acid, nicotine, pantothenic acid, vitamin B6, vitamin B1 and riboflavin;

[0142] 3) 0.4% by mass fraction of NaCl and 0.4% by mass fraction of KCl,

[0143] 4) 5% by mass fraction of insulin and 5% by mass fraction of transferrin; and

[0144] 5) 2.2% by mass fraction of glucose, 1.5% by mass fraction of penicillin and 1.5% by mass fraction of streptomycin.

[0145] One skilled in the art will understand that the embodiment of the present invention as shown in the drawings and described above is exemplary only and not intended to be limiting.

[0146] It will thus be seen that the objects of the present invention have been fully and effectively accomplished. Its embodiments have been shown and described for the purposes of illustrating the functional and structural principles of the present invention and is subject to change without departure from such principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the following claims.