CN113136411A - Evaluation method using human stem cells, drug evaluation method, and detection kit - Google Patents
Evaluation method using human stem cells, drug evaluation method, and detection kit Download PDFInfo
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Abstract
The present invention relates to an evaluation method using human stem cells, a drug evaluation method, and a detection kit using human stem cells. The evaluation method using human stem cells, comprising the steps of: (1) providing in vitro stem cells; (2) adding a substance to be tested to act on the in-vitro stem cells in the step (1); (3) detecting the condition of the in vitro stem cells after the step (2) is finished; (4) and (4) evaluating the influence of the substance to be detected on the stem cells according to the condition of the in-vitro stem cells detected in the step (3). The method for evaluating the human stem cells, the method for evaluating the drugs by using the human stem cells and the detection kit by using the human stem cells can establish a quantitative, rapid and low-cost in-vitro detection platform and an evaluation system, can replace animal experiment detection, and provide a detection basis of a stem cell layer for in-vitro detection of a human body.
Description
Technical Field
The present invention relates to an evaluation method using human stem cells, a drug evaluation method using human stem cells, and a detection kit using human stem cells.
Background
The new medicine needs to go through four processes of time consumption and money consumption in the clinical stage of research, screening-preclinical research and new medicine application approval on the market after being researched and developed. Generally, the time is more than 10 years, and hundreds of millions or even billions of dollars are spent. The main contributing factors to the success of a new drug are the determination of drug toxicity and efficacy at the preclinical experimental stage. Early accurate and effective toxicity determination is critical to ensuring the success of a drug.
At present, pharmacological and toxicological action evaluation of drugs in preclinical experimental stages is mainly developed through animal level, and the current animal determination system for analyzing potential drug molecular toxicity effects has considerable limitations, not only needs a large number of animals, but also is time-consuming and huge, generally needs 2-4 years, is derived from toxicity test results of animals, and cannot accurately represent human body reactions and is difficult to standardize. Partial pharmacological and toxicological action evaluation is also developed from the human cell level, but the measurement indexes are only limited to individual organs or tissues of the human body, and the system and the comprehensiveness are lacked.
Disclosure of Invention
It is an object of the present invention to overcome the disadvantages of the prior art and to provide a method for evaluating human stem cells, a method for evaluating a drug using human stem cells, and a test kit using human stem cells.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
an evaluation method using human stem cells, comprising the steps of: (1) providing in vitro stem cells; (2) adding a substance to be tested to act on the in-vitro stem cells in the step (1); (3) detecting the condition of the in vitro stem cells after the step (2) is finished; (4) and (4) evaluating the influence of the substance to be detected on the stem cells according to the condition of the in-vitro stem cells detected in the step (3).
According to one embodiment of the present invention, in the step (1), the in vitro stem cells are obtained by isolating from human organ tissues and amplifying in vitro.
According to one embodiment of the invention, the stem cells comprise embryonic stem cells and/or adult stem cells.
According to one technical scheme of the invention, the adult stem cells comprise one or more of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells.
According to one embodiment of the present invention, in the step (2), the test substance is allowed to act on a plurality of and/or a plurality of stem cells in each of the plurality of and/or the plurality of stem cells under the same condition.
According to one embodiment of the present invention, in the step (2), the substance to be tested is allowed to act on one or more in vitro stem cells selected from the group consisting of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells, and skin tissue stem cells under the same conditions.
According to one embodiment of the present invention, the step (2) further comprises embryonic stem cells.
According to one of the technical solutions of the present invention, the same conditions include the same temperature, the same action time, the same concentration of the substance to be measured, the same contact manner, the same ratio, the same generation of in vitro stem cells, the same oxygen concentration, the same carbon dioxide concentration, the same action frequency, and the same repetition number.
According to one embodiment of the present invention, in the step (2), the same test substance is allowed to act on the same stem cell in vitro under different conditions.
According to one embodiment of the present invention, in the step (3), the mortality and/or metabolic rate of the in vitro stem cells is detected; the effect of the test substance on the stem cells was evaluated based on comparison of data before and after the test substance was used.
According to one embodiment of the present invention, the status of the in vitro stem cells is detected a plurality of times by repeating the step (3) at different times.
According to one embodiment of the present invention, in step (1), before starting step (2), the condition of the in vitro stem cells is detected; in the step (4), the detection result of the in vitro stem cells in the step (1) is compared with the detection result in the step (3), and the influence of the substance to be detected on the in vitro stem cells is evaluated.
A method for evaluating a drug using human stem cells, comprising the steps of: (1) providing in vitro stem cells; (2) adding a drug to be detected to enable the drug to be detected to act on the in-vitro stem cells in the step (1); (3) detecting the condition of the in vitro stem cells after the step (2) is finished; (4) and (4) evaluating the influence of the medicament on the stem cells according to the condition of the in-vitro stem cells detected in the step (3).
According to one embodiment of the present invention, the drug efficacy test is performed under the same conditions as those of the steps (1) to (4).
According to one embodiment of the present invention, in the drug efficacy test, the drug to be tested is allowed to act on cells or pathogenic factors of the selected disease under the same conditions, and then the condition of the cells or pathogenic factors of the selected disease is detected.
According to one embodiment of the present invention, the cells with the selected disease are stem cells or functional cells with the selected disease; the pathogenic agent comprises one or more of virus, fungus or bacteria.
According to one embodiment of the present invention, in step (1), cells or pathogenic factors of a selected disease are also provided; in the step (2), adding a drug to be tested to the cells or pathogenic factors with the selected diseases in the step (1); in the step (3), detecting the condition of the cells or pathogenic factors with the selected diseases after the step (2); in the step (4), the effectiveness of the medicament is evaluated according to the result of the detection in the step (3).
According to one embodiment of the present invention, in the step (1), the stem cells provided are stem cells not suffering from the selected disease; evaluating the toxicity of the drug or selected component of the drug by steps (1) through (4).
According to one embodiment of the present invention, in the step (1), the in vitro stem cells are obtained by isolating from human organ tissues and amplifying in vitro.
According to one embodiment of the invention, the in vitro stem cells comprise embryonic stem cells and/or adult stem cells.
According to one technical scheme of the invention, the adult stem cells comprise one or more of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells.
According to one embodiment of the present invention, in the step (2), the test substance is allowed to act on a plurality of and/or a plurality of stem cells in each of the plurality of and/or the plurality of stem cells under the same condition.
According to one of the technical schemes of the present invention, in the step (2), under the same condition, the drug to be tested or the selected component of the drug to be tested is allowed to act on one or more in vitro stem cells of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells at the same time.
According to one embodiment of the present invention, the step (2) further comprises embryonic stem cells.
According to one of the technical schemes of the present invention, the same conditions include the same temperature, the same action time, the same concentration of the drug to be tested, the same contact manner, the same ratio, the same generation of in vitro stem cells, the same oxygen concentration, the same carbon dioxide concentration, the same action frequency, and the same repetition number.
According to one of the technical schemes of the invention, in the step (2), the same drug to be tested is allowed to act on the stem cells in vitro under different conditions for the same stem cell.
According to one embodiment of the present invention, in the step (3), the mortality and/or metabolic rate of the in vitro stem cells is detected; the effect of the drug on the stem cells is assessed by comparing the data before and after use of the drug or selected components of the drug.
According to one embodiment of the present invention, the status of the extracorporeal stem cells is detected a plurality of times by repeating the step (3) at different times.
According to one embodiment of the present invention, in step (1), before starting step (2), the condition of the in vitro stem cells is detected; in the step (4), the detection result of the in-vitro stem cells in the step (1) is compared with the detection result in the step (3), and the influence of the drug to be detected on the in-vitro stem cells is evaluated.
A detection kit using human stem cells is characterized by comprising a box body, wherein the box body is filled with in-vitro stem cells; the in vitro stem cells are obtained by separating and amplifying in vitro organs and tissues of a human body; the in vitro stem cells comprise one or more of adult stem cells and/or embryonic stem cells.
According to one technical scheme of the invention, the adult stem cells comprise one or more of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells.
According to one technical scheme of the invention, the stem cells comprise a plurality of types, and the plurality of types of stem cells are respectively and independently contained.
The method for evaluating the human stem cells, the method for evaluating the drugs by using the human stem cells and the detection kit by using the human stem cells can establish a quantitative, rapid and low-cost in-vitro detection platform and an evaluation system, can replace animal experiment detection, and provide a detection basis of a stem cell layer for in-vitro detection of a human body; the detection kit using the human stem cells can contain the stem cells of the human main system, and has wide application range and unlimited market prospect.
The stem cell evaluation method and the kit can be used for drug safety and efficacy detection evaluation, food safety and efficacy detection evaluation, cosmetic safety and efficacy detection evaluation, potential environmental toxicant detection evaluation, tea safety and efficacy detection evaluation, tobacco safety detection evaluation and the like.
The invention also has the following advantages:
(1) the system contains adult stem cells of main organ tissues of all systems of the whole body, so that the system can comprehensively detect and evaluate the reaction of all main tissue cells of the body to target molecules or preparations to be detected.
(2) The system adopts a high-content screening (HCS) technology combining an automatic microscope and quantitative image analysis to quantitatively analyze the change of stem cells in different indexes such as morphology, growth, differentiation, physiology, metabolism and the like under the same condition and in the same time period, and detects the reaction of each system of the whole body to the condition to be detected.
(3) And standard property, in the system, the reaction of organs and tissues of each large system of the whole body to molecules to be detected is carried out under standard conditions, so that the uniformity and the standard property are realized.
(4) In the system, the reaction of organs and tissues of each large system of the whole body from a human body to molecules to be detected is carried out under the same condition, and all data are direct reaction of human cells, but not interference from other species sources, so that the system has the highest accuracy.
(5) The system can be used for making different combinations of main organ tissue stem cells of different systems according to detection requirements.
(6) And the expandability is that the obtained stem cells of organs and tissues of various systems of the whole body of the human body in the system can be applied to various aspects such as tissue engineering and the like.
Drawings
FIG. 1 is a flowchart of an evaluation method using human stem cells in the present invention.
Detailed Description
As shown in fig. 1, step (1): separating stem cells from human organ tissues and carrying out in-vitro amplification; establishing an in-vitro stem cell bank covering each system tissue of a human body.
The stem cells include embryonic stem cells and/or adult stem cells. The particular stem cell may be selected as required for the assay.
Such as circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells, and stem cell in vitro systems of skin tissue stem cells and embryonic stem cells. The circulatory stem cells include but are not limited to cardiac stem cells, vascular stem cells and bone marrow stem cells; the digestive system stem cells include, but are not limited to, esophageal stem cells, gastric stem cells, large intestinal stem cells, small intestinal stem cells, and liver stem cells; the neural stem cells include, but are not limited to, neural stem cells; the immune system stem cells include, but are not limited to, bone marrow mesenchymal stem cells; the endocrine system stem cells include, but are not limited to, ovarian stem cells, testicular stem cells, and adrenal stem cells; the reproductive system stem cells include, but are not limited to, uterine stem cells, ovarian stem cells, and testicular stem cells; the skeletal system ovarian stem cells and testicular stem cells include but are not limited to skeletal muscle ovarian stem cells and testicular stem cells; urinary system stem cells include, but are not limited to, kidney stem cells and prostate stem cells; the muscle stem cells include but are not limited to smooth muscle stem cells, skeletal muscle stem cells, cardiac muscle stem cells; the skin tissue stem cells include, but are not limited to, dermal stem cells, epidermal stem cells, hair follicle stem cells, and sweat gland stem cells.
The collection and amplification of in vitro stem cells can adopt conventional technical means, and can also adopt the following methods in the invention:
tissue collection:
1. according to the requirements of national ethical management, after completing the informed consent, corresponding tissues are obtained by operation or biopsy (taking the tissues from the body) in hospitals;
2. simply washing the obtained tissue with normal-temperature normal saline to remove extravasated blood on the tissue;
3. storing the cleaned tissue in a sealed test tube containing a sterile tissue preservation buffer;
4. putting the mixture into a low-temperature ice box at the temperature of 4-8 ℃;
5. sending to a laboratory;
stem cell isolation:
1. aseptically processing the sealed test tube containing the obtained tissue block, and placing the test tube into a biological safety cabinet;
2. taking out from the tissue preservation solution (normal saline) by using forceps;
3. placing the tissue into iodine tincture, and fully soaking for 1 minute at room temperature;
4. placing the tissue into 75% alcohol, fully washing for 3 minutes at room temperature;
5. washing: phosphate buffer, room temperature, 3 minutes/time, washing 3 times;
6. shearing the harvested tissue with a vertical mechanical force;
7. transfer it into a 15 or 50 ml conical tube (depending on the volume of the sample);
8. adding appropriate amount of tissue enzymolysis enzyme solution, covering tissue, enzyme treating at 37 deg.C for 1 hr, and shaking 1 time every 3 min;
9. enzyme inactivation: adding a proper amount of fetal calf serum to stop the enzymolysis reaction;
10. and (3) filtering: filtering through 100 μm and 40 μm filter membranes respectively;
11. centrifuging 1: 360g,8 min, 4 ℃, the supernatant was carefully collected;
12. adding a proper amount of tissue enzymolysis enzyme solution, and suspending the obtained cells;
13. counting 1: taking 10 microliter of cell suspension, staining by trypan blue and counting;
14. centrifuging 2: 360g,8 minutes, 4 ℃, carefully discarding the supernatant;
15. counting 2: suspending cells in 1ml of cell culture solution, taking 10 microliter of cell suspension, staining by trypan blue and counting;
stem cell culture and passage:
1. suspending the tissue stem cells isolated by the above procedure in a stem cell culture solution;
2. transferring the seeds into a cell culture plate;
3. culturing in cell culture box at 37 deg.C under 5% CO2 and saturated humidity;
4. examining the cultured cells daily with an inverted phase contrast microscope;
5. when the cells grew to 80% coverage, the cells were passaged as follows;
6. the growth medium was aspirated away and the cells were washed twice with phosphate buffer;
7. aspirate all phosphate buffer and add 2 ml of 37 ℃ pre-warmed trypsin;
8. putting the culture dish into a carbon dioxide incubator for 3-5 minutes;
9. add 8 ml growth medium and collect all cells in a 12 ml tube;
10. centrifuging at 5000 rpm for 5min at room temperature;
11. sucking out supernatant, suspending cells by using phosphate buffer, and centrifuging for 5 minutes at the rotating speed of 5000 rpm at room temperature;
this washing step was repeated twice and the cells were counted using a hemocytometer;
12. suspending the cells in growth medium and gently buffering the cells with a pipette several times to ensure even distribution of the cells;
13. subculturing the cells with growth medium at a density of 2x 103 cells per square centimeter;
14. these are first generation (p1) cells;
15. cells were examined daily, medium was changed every 72 hours, cells were passaged at 90% confluence;
purifying stem cells:
obtaining purified target tissue stem cells by using a flow cytometer;
1. from the stem cells isolated and cultured by the above procedure, a single cell suspension for cell sorting was prepared
2. Taking out the culture tray and putting the culture tray into a biological safety cabinet;
3. the culture solution is aspirated;
4. washing the stem cells in the three-time culture with a phosphate buffer;
5. treating the cells with trypsin to detach them from the walls;
6. adding a proper amount of fetal calf serum to stop the enzymolysis reaction;
7. washing the cells with a buffer solution special for staining for 3 times;
8. centrifuging, removing supernatant, and harvesting cells;
9. suspending the obtained cells with a special immunostaining blocker;
10. adding the target antibody for purification (appropriate amount of concentration);
11. culturing the cells in the dark at 4 ℃ for 30-60 minutes;
12. adding;
13. purifying and sorting to obtain a second target antibody, and repeating the steps 7-9;
14. suspending the stained cells with a sorting-specific sorting buffer to a final concentration of about 4x 106/ml;
15. prior to sorting, cells were passed through a 40 μm filter to remove non-single cells;
16. collecting target cells obtained by sorting through a flow cytometer;
17. transforming the obtained purified stem cells into a culture disc;
18. culturing in cell culture box at 37 deg.C under 5% CO2 and saturated humidity;
and (3) stem cell identification:
A. genotype identification, phenotype identification, growth identification and karyotype identification.
I. Identification of stem cell characteristic genes
RNA extraction and reverse transcription-PCR;
1. extracting total RNA from the isolated or expanded stem cells using Tripure isolation kit from Roche (according to manufacturer's instructions);
2. cDNA was synthesized by reverse transcription using the Improm reverse transcription system from Promega (according to the manufacturer's instructions);
3. amplifying the characteristic gene of the target stem cell by using a PCR (polymerase chain reaction) amplification instrument according to the corresponding characteristic gene primer of the target stem cell;
characterization of Stem cell proliferation
The growth kinetics of stem cells isolated or expanded from the target tissue in primary culture were evaluated using a cell proliferation assay (biomedicine) (P0).
Primary culture cells of different origins (p < 0.01) were repeatedly seeded at 1.0X 103/cm2 in t 75 dishes to observe the expansion of mscs in culture. The medium was changed every 3 days. When the cultures reached 90% confluence, both sources of adherent layers were washed with PBS to remove residual media and cells were harvested with 0.05% trypsin EDTA at 37 ℃ for 5-10 min. At the end of each passage, the process was repeated at passage 1, 2, 3 and 4, and the cells were counted repeatedly using a hemocytometer. The total number of proliferations in the culture after 4 passages was estimated and the percentage increase of the total number of stem cells of the target tissue was calculated.
Identification of Stem cell clonogenic units
The self-renewal (growth, proliferation) ability of stem cells of the target tissue was evaluated by Colony-forming unit method (Colony-forming units-stem cell assay):
in a 60mm (28 cm-2) tissue culture dish (bd falcon)TM) In (2) inoculating primary culture cells (p < 0.01) from different sources into a basal medium at a speed of 1.0X 102 cells/cm-2. The medium was changed every 3 days. After incubation at 37 ℃ in 5% humidified carbon dioxide for 14 days, the medium was removed, the cells were washed with PBS, fixed with methanol, and stained with May Gr nwald Giemsa (Merck-Kgaa). A cell cluster containing 50 or more cells was used as a CFU-SC colony, and the cloning efficiency was calculated by (number of colonies/number of inoculated cells) × 100.
Identification of telomere length of stem cells in target tissue
The length of the genome DNA telomerase of the target tissue stem cells of different culture generations is determined by a reagent system special for Roche.
Analysis of target tissue Stem cell karyotype
The stem cells of the target tissue to be tested were treated with colchicine (100ng/ml, Roche) for 20 minutes. Then the cells were detached with accutase enzyme (Invitrogen), collected, and centrifuged; gently suspend the cells in hypotonic solution, fix the cells with mixed solution (3:1v/v methanol: glacial acetic acid); the fixed cells were dropped on a clean wet slide, air-dried, and then stained with gimbah staining solution (giemsa). And (5) observing by using a microscope, and recording the chromosome karyotype.
Identification of stem cell immunophenotype in target tissue
1. Taking out the culture tray and putting the culture tray into a biological safety cabinet;
2. the culture solution is aspirated;
3. washing the stem cells in the three-time culture with a phosphate buffer;
4. treating the cells with trypsin to detach them from the walls;
5. adding a proper amount of fetal calf serum to stop the enzymolysis reaction;
6. washing the cells with buffer solution 3 times;
7. centrifuging, removing supernatant, and harvesting cells;
8. suspending the cells with a buffer containing 5% bovine serum, centrifuging, and repeatedly washing twice, and then suspending the cells in a buffer containing 5% bovine serum;
9. counting cells, aliquoting 5 × 105 cells in 12 ml tubes;
10. centrifuging, removing supernatant, and harvesting cells;
11. carefully suspend cells 5 × 105 cells in 90 μ l of 5% bovine serum buffer; transferring it to a 1.5 ml centrifuge tube;
12. add 10. mu.l antibody/isotype control to 5X 105 cells;
13. shake on ice away from light for 1 hour;
14. centrifuge at 800rcf for 5 minutes;
15. suspending the cells in a buffer of 5% bovine serum;
16. repeating the washing step two to three times;
17. suspending cells each sample in 500 microliters of 5% bovine serum buffer;
18. analyzing live cells using forward scatter and side scatter flow cytometry;
19. the target tissue stem cell group immunophenotype should be positive for CD29, CD44, CD105, CD73, CD166, and Sca-1, negative for CD11b, CD14, CD45, and CD 31;
immunofluorescence analysis of Stem cells of target tissue
Target tissue stem cells were fixed with freshly prepared 4% (w/v) paraformaldehyde for 20 minutes at room temperature, washed with PBS, and labeled with the following antibodies: NG2(1:100, Merck Millipore, UK), platelet-derived growth factor receptor-beta (PDgfr-beta) (1:50, Santa Cruz, UK), Vimentin (1:100, Abcam, UK), GATA-4(1:50, Santa Cruz), Oct-4(1:400, Abuk), SOX-2(1:100, Merck Margarobo) and C-Kit (1:40, David, UK), alpha-smooth muscle actin (1:100, koda), alpha-smooth muscle (1:50, Abcam), alpha-non-muscle myosin iiib (1:500, Abcam), alpha-smooth muscle myosin heavy chain 11(1:50, Abcam), alpha-retinol binding protein 1(1:100, Abcam) and alpha-calpain (1:100, Abcam), Capra-dhcam (1:50, VEGFR-1: 50, Cruz), cell signaling, UK) and von-Willebrand factor (1:50, DAKO, UK), cardiomyocyte properties: α -sarcomeric actin (1:500, Sigma Aldrich, UK) and connexin 43(1:40, san Cruis). To detect intracellular antigens, cells were permeabilized with 0.1% (v/v) Triton X100(Sigma-Aldrich) diluted in PBS for 10 minutes at room temperature, the first antibody was incubated for 16 hours at 4 ℃ and for 2 hours at 37 ℃ except for c-kit and gap junction protein 43. Secondary antibodies were incubated on the cells for 1 hour at 20 ℃ (1:200 anti-rabbit Alexa 488, 1:200 anti-mouse Alexa 488, or Alexa 568[ Life Technologies, uk ]) in the dark. Nuclei were counterstained with 4', 6-diamino-2-phenylindole (dapi) (sigma-aldrich). Slides were fixed with fluorine-g (sigma-aldrich). Cells were analyzed at 400 x magnification. All experiments were performed in triplicate using Adobe Photoshop software to synthesize and overlay images (Adobe), and 3-7 cell lines were evaluated.
B. Differentiation capacity identification of stem cells through cartilage tissue differentiation, bone tissue differentiation and adipose tissue differentiation experiments
I. Inducing differentiation of adipose tissues;
1. preparing a fat-forming induction culture medium and a maintenance culture medium. This 2 medium was stored in the dark at 4 ℃ until the fat induction culture period.
2. The target tissue stem cells were seeded into 24-well plates at a density of 2 × 104 viable cells per well in 500 μ l growth medium. The dishes were incubated overnight at 37 ℃ under 5% carbon dioxide to allow the cells to adhere;
3. the growth medium was aspirated off, 500 μ L of adipogenic induction medium was added to each well, incubated at 37 ℃ for 3 days with 5% carbon dioxide, and then this alternating cycle of induction/maintenance medium was repeated twice (an additional 12 days) again with 3 days of 500 μ L of adipose maintenance medium;
4. after 18 days of the induction/maintenance cycle, the cultures were refreshed with adipose maintenance medium for 3 days. The total days of adipogenic culture was 21 days. Observing the change of the culture under an inverted microscope to observe the differentiation process;
5. after the adipogenic culture for 21 days, sucking the culture medium, washing with 500 mu L PBS, adding 500 mu L10% neutral formalin buffer solution, and keeping the temperature at room temperature for 30 min;
6. absorbing formalin buffer solution, and washing with PBS twice;
7. adding 500 mu L of oil red O working staining solution, and keeping the temperature at room temperature for 50 minutes;
8. the staining solution was aspirated, washed three times with 500. mu.L PBS, and counterstained with 500. mu.L hemoflavin solution for 5 minutes;
9. examination under an inverted microscope and recording the stained culture;
bone tissue induced differentiation
1. Preparing an osteogenic tissue induction culture medium. Storing the culture medium in the dark at 4 deg.C until the osteogenic tissue induction culture period;
2. the target tissue stem cells were seeded into 24-well plates at a density of 2 × 104 viable cells per well in 500 μ l growth medium. The dishes were incubated overnight at 37 ℃ under 5% carbon dioxide to allow the cells to adhere;
3. taking out the CCM, adding 500 mu L osteogenic tissue induction culture medium, and putting the culture plate back to the incubator;
4. the whole osteogenic tissue induction medium was renewed every 3 days for 21 days. Observing the change of the culture with an inverted microscope;
5. after the osteogenic tissue is subjected to induction culture for 21 days, sucking the culture medium, washing with 500 mu L PBS, adding 500 mu L10% neutral formalin buffer solution, and carrying out room temperature 30 min;
6. formalin buffer was discarded and washed twice with PBS. Adding 500 mul of alkaline phosphatase staining solution, and incubating for 30-45 minutes at room temperature to form staining;
7. the staining solution was discarded and washed three times with PBS. The alkaline phosphatase is positively stained, and the cell membrane is pink to red;
8. to detect confocal of early and late osteogenic markers (alkaline phosphatase and calcium phosphate minerals, respectively), PBS was removed from alkaline phosphatase stained wells and 500 μ L von Kossa stain (2.5% AgNO3) was added in the dark for 30 min;
9. discarding the solution and rinsing twice with double distilled water;
10. checking by an inverted microscope, and recording a dyeing result;
induced differentiation of cartilage tissue
1. For chondrocyte differentiation, target tissue stem cells were diluted to a final concentration of 2.5 × 105 cells/mL DMEM;
2. transfer to a 15mL polypropylene conical tube with a total volume of 1mL and centrifuge at 150g × 5min at room temperature to completely aspirate the medium;
3. adding 1mL preheated cartilage tissue induction culture medium, suspending cells, centrifuging, repeating for 2 times, culturing at 37 deg.C in humidified air of 5% CO2, and culturing in 1mL chondrogenesis culture medium;
4. the cartilage tissue induction culture medium is replaced every 3 days;
5. after 24 days of culture, the pellets were embedded in paraffin;
6. cutting into 5mm slices;
7. detecting the matrix rich in proteoglycan in the section by using an Alcian blue staining;
8. performing immunofluorescence detection on the type II collagen and a secondary anti-mouse antibody (both from Chemicon International) combined with FITC by using a mouse anti-human type II collagen monoclonal antibody to prove that chondrocyte differentiation is performed;
9. inverted microscopy and staining results recorded.
Step (2): according to the detection requirement, selecting a corresponding human tissue stem cell model and adding the human tissue stem cell model into the detection kit to form the detection kit using the human stem cells.
When only the influence of the substance to be detected on the stem cells needs to be detected, healthy multiple and/or multiple in vitro stem cells are selected for simultaneous detection. For example, the effect of a certain cosmetic on human stem cells can be detected by simultaneously selecting circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells. And selecting a plurality of stem cells for each stem cell to form a detection kit. Of course, embryonic stem cells may also be included in the kit. The specific number of each stem cell can be determined according to the actual experimental needs.
Adding quantitative substance to be detected into the detection kit, wherein the substance to be detected is a substance to be detected, and can be cosmetics, tobacco products, medicines and the like. The substance to be tested may be either in the form of the final product of the substance to be tested or may be a selected characteristic component. For example, the substance to be tested is a cosmetic, and can be a finished product of cosmetics sold on the market, which comprises auxiliary components such as a substrate, a preservative, a pigment, an essence and the like, or can be a main component of the cosmetics, such as the substrate. The substance to be detected is a medicine, and can be a finished product medicine which is sold in the market and comprises auxiliary materials, and can also be a main functional component or ingredient of the medicine. For example, for the capsule medicine, the capsule medicine can be added integrally; it is also possible to add only the pharmaceutical powder in the capsule, or even only the chemical components that could potentially have a therapeutic effect on the selected disease.
The substance to be detected acts on a plurality of in vitro stem cells in the kit at the same time. The mode of action can be determined according to the substance to be detected, for example, if the substance to be detected is a gas, the stem cells or the stem cell culture solution are exposed to the gas to be detected, if the substance to be detected is a liquid or a solid, the solution containing the substance to be detected is added to the stem cell-containing culture solution to contact the stem cells.
The effect of the test substance on various stem cells can be performed under the same condition or different conditions. The same conditions include, but are not limited to, the same temperature, the same action time, the same concentration of the substance to be tested, the same contact manner, the same ratio, the same generation of in vitro stem cells, the same oxygen concentration, the same carbon dioxide concentration, the same action frequency, and the same repetition number. The specific selection condition can be selected according to actual conditions.
Preparation of the kit:
the method comprises the following basic steps:
1. respectively suspending target tissue stem cells from different sources by using growth culture media to prepare single cell suspensions with the same concentration;
2. cell culture plates (96-well plates, 384-well plates) required for labeling assays;
3. uniformly marking and adding the prepared target tissue stem cell single cell suspension into a disc according to equal quantity;
4. six wells were added for each tissue cell;
5. placing the culture dish in a cell culture box, culturing at 37 ℃ and 5% CO2 under saturated humidity overnight to allow the cells to adhere;
after 6.48 hours, or directly loading the sample to carry out a drug molecule screening test, or;
7. taking the cell culture disc out of the incubator;
8. absorbing and discarding the culture solution;
9. washing twice with pre-warmed phosphoric acid buffer;
10. adding a proper amount of fresh cell freezing solution;
11. putting into a refrigerator with the temperature of 20 ℃ below zero, and preserving for 12-18 hours;
12. transferring into a refrigerator at 80 ℃ below zero for standby;
sample adding:
1. preparing a target molecule solution to be detected according to the requirement required by the detection;
2. directly homogenizing the molecular liquid to be detected by a manual or mechanical method, and adding the molecular liquid to each hole in equal quantity;
3. transferring the plate into a cell culture box, and arranging an incubation time course according to specific measurement requirements;
4. after the expected reaction time course is reached, taking the disc out of the cell culture box, and starting the subsequent required measurement steps;
and (4): after a proper reaction time, the detection kit in the step 3 is detected, and the reaction (change of death rate, metabolic rate and the like) of different adult stem cells existing in the test system to the molecules/drugs to be detected is quantitatively determined under the same condition.
Step (3), measuring; detecting the condition of the stem cells completing the step (2), wherein the detected data can be various data for evaluating the health condition of the stem cells, such as the death rate and/or the metabolic rate of the stem cells and the like.
Step (4), evaluating; and (4) forming a quantitative detection and evaluation result by using the data obtained by the detection in the step (3). Comparing the detection result of the stem cells subjected to the step (2) with the detection result of the stem cells not subjected to the step (2), and evaluating the effect of the substance to be tested in the step (2) on the in vitro stem cells. The comparison object can be the detection data of the stem cells provided in the step (1) before being affected by the substance to be detected or the accepted theoretical data. The evaluation can evaluate the influence of the same substance on different stem cells and can also evaluate the influence of the same substance on the same stem cells under different conditions.
MTT assessment of the cytotoxic Effect of test molecules on cultured cells by measuring the changes in the number of induced cell deaths
1. Appropriate numbers of cells were harvested, counted and plated in 100 μ L complete medium;
2. adding 10pL of a drug solution with the concentration 10 times that of the required concentration;
3. culturing at 37 deg.C and 5% carbon dioxide in humidified environment for different time;
4. 10fl MTT solution (5mg/ml) was added;
5. culturing in 5% carbon dioxide cell culture box at 37 deg.C for 4-6 hr;
6. centrifuging at 2000 rpm for 10 min;
7. taking out the culture medium;
8. adding 100 mu L of dimethyl sulfoxide;
9. mechanically stirring the plate until the praline crystals are dissolved;
10. reading the optical density of the micro-culture plate at the test wavelength of 550 nm and the reference wavelength of 660 nm respectively;
11. calculating the average value of optical density of different repeated quantities of the same sample;
12. evaluating the percentage of each value relative to a control value;
13. plotting optical density as a percentage of drug concentration in a semi-logarithmic graph;
14. ID50 was determined from the dose-response curve.
The toxicity of the test substance can be verified when the healthy in vitro stem cells used in the aforementioned steps (1) to (4) are used.
For drug research, drug toxicity detection and drug effectiveness detection can be carried out simultaneously. Preferably, the drug efficacy test and the drug toxicity test are performed simultaneously under the same conditions, that is, the conditions under which the drug to be tested acts on the in vitro stem cells are the same as the conditions under which the drug to be tested acts on the cells with the selected disease or the pathogenic factors; then the influence of the drug on the in vitro stem cells and the influence of the cells or pathogenic factors with the selected diseases are respectively detected and evaluated, and the evaluation results of the drug effectiveness and toxicity are obtained.
The drug toxicity test is preferably derived from stem cells of a healthy human, at least from stem cells of a human not suffering from the selected disease. For example, when detecting the toxicity of a drug for treating liver cancer, the in vitro stem cells used in the toxicity detection are at least those of a human body not suffering from liver cancer. More preferably, stem cells derived from a completely healthy human body are collected.
The detection of the effectiveness of the drug may be performed on cells with the selected disease or on pathogenic agents. The selected disease is determined according to actual detection requirements, such as cells with benign tumor, cancer, hepatitis, tissue hyperplasia, etc. The cells can be either stem cells with a selected disease, such as tumor stem cells, or functional cells with a selected disease, hepatocytes with hepatitis; but also other cells which are affected by pathogenic agents and which present a disease state. Of course, the drug to be tested may also be allowed to act directly on pathogenic agents such as viruses, fungi or bacteria.
The effectiveness of the drug can also be tested by other methods known in the art, provided that it is tested under the same conditions as the toxicity test on stem cells in vitro.
The invention also provides a detection kit using the human stem cells, which structurally comprises a box body, wherein the box body is internally filled with the in-vitro stem cells; the in vitro stem cells comprise one or more of adult stem cells and/or embryonic stem cells, and preferably comprise the adult stem cells and the embryonic stem cells. The adult stem cells comprise one or more of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells; preferably all are included. The plurality of stem cells are respectively and independently contained, and the number of each stem cell is multiple. The shape and structure of the box body can adopt the existing conventional box body as long as the box body can divide various stem cells and is suitable for putting in the measured substances.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, and any modifications, equivalents or improvements that are within the spirit of the present invention are intended to be covered by the following claims.
Claims (32)
1. An evaluation method using human stem cells, comprising the steps of: (1) providing in vitro stem cells; (2) adding a substance to be tested to act on the in-vitro stem cells in the step (1); (3) detecting the condition of the in vitro stem cells after the step (2) is finished; (4) and (4) evaluating the influence of the substance to be detected on the stem cells according to the condition of the in-vitro stem cells detected in the step (3).
2. The method for evaluating human stem cells according to claim 1, wherein in step (1), the in vitro stem cells are obtained by in vitro amplification isolated from human organ tissue.
3. The evaluation method according to claim 1, wherein the stem cells comprise embryonic stem cells and/or adult stem cells.
4. The method according to claim 3, wherein the adult stem cells include one or more of circulatory stem cells, digestive stem cells, respiratory stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells, and skin tissue stem cells.
5. The method for evaluating human stem cells according to claim 1, wherein in the step (2), the test substance is allowed to act on a plurality of and/or a plurality of stem cells in each under the same condition.
6. The method for evaluating an antibody according to claim 1 or 5, wherein in said step (2), said test substance is allowed to act simultaneously on one or more in vitro stem cells selected from the group consisting of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells under the same conditions.
7. The evaluation method according to claim 6, wherein said step (2) further comprises an embryonic stem cell.
8. The method for evaluating human stem cells according to claim 5, wherein the same conditions include the same temperature, the same action time, the same concentration of the test substance, the same contact pattern, the same ratio, the same generation of in vitro stem cells, the same concentration of oxygen, the same concentration of carbon dioxide, the same action frequency, and the same number of repetitions.
9. The method for evaluating human stem cells according to claim 1, wherein in said step (2), the same test substance is allowed to act on the same kind of stem cells in vitro under different conditions.
10. The evaluation method according to claim 1, wherein in the step (3), the mortality and/or metabolic rate of the in vitro stem cells is detected; the effect of the test substance on the stem cells was evaluated based on comparison of data before and after the test substance was used.
11. The assessment method according to claim 1, wherein said step (3) is repeated at different times to detect the condition of the in vitro stem cells a plurality of times.
12. The method for evaluating using human stem cells according to claim 1, wherein in step (1), before starting step (2), the condition of the in vitro stem cells is detected; in the step (4), the detection result of the in vitro stem cells in the step (1) is compared with the detection result in the step (3), and the influence of the substance to be detected on the in vitro stem cells is evaluated.
13. A method for evaluating a drug using human stem cells, comprising the steps of: (1) providing in vitro stem cells; (2) adding a drug to be detected to enable the drug to be detected to act on the in-vitro stem cells in the step (1); (3) detecting the condition of the in vitro stem cells after the step (2) is finished; (4) and (4) evaluating the influence of the medicament on the stem cells according to the condition of the in-vitro stem cells detected in the step (3).
14. The method for evaluating a drug using human stem cells according to claim 13, wherein the drug efficacy test is conducted under the same conditions as in steps (1) to (4).
15. The method of claim 14, wherein the test drug is allowed to act on cells or pathogenic agents of the selected disease under the same conditions in the drug efficacy test, and then the condition of the cells or pathogenic agents of the selected disease is examined.
16. The method for evaluating a drug using human stem cells according to claim 15, wherein the cells having the selected disease are stem cells or functional cells having the selected disease; the pathogenic agent comprises one or more of virus, fungus or bacteria.
17. The method for evaluating a drug using human stem cells according to claim 14, 15 or 16, wherein in the step (1), cells or pathogenic agents having a selected disease are further provided; in the step (2), adding a drug to be tested to the cells or pathogenic factors with the selected diseases in the step (1); in the step (3), detecting the condition of the cells or pathogenic factors with the selected diseases after the step (2); in the step (4), the effectiveness of the medicament is evaluated according to the result of the detection in the step (3).
18. The method for evaluating a drug using human stem cells according to claim 13, wherein in the step (1), the stem cells provided are stem cells not suffering from the selected disease; evaluating the toxicity of the drug or selected component of the drug by steps (1) through (4).
19. The method for evaluating a drug using human stem cells according to claim 13, wherein in step (1), the in vitro stem cells are obtained by in vitro amplification isolated from human organ tissues.
20. The method for drug evaluation according to claim 13, wherein the in vitro stem cells include the stem cells including embryonic stem cells and/or adult stem cells.
21. The method of claim 20, wherein the adult stem cells comprise one or more of circulatory stem cells, digestive stem cells, respiratory stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells, and skin tissue stem cells.
22. The method for evaluating a drug using human stem cells according to claim 13, wherein in the step (2), the test substance is allowed to act on a plurality of and/or a plurality of stem cells in each under the same condition.
23. The method for evaluating a drug using human stem cells according to claim 22, wherein in the step (2), the drug to be tested or the selected component of the drug to be tested is allowed to act simultaneously on one or more in vitro stem cells selected from the group consisting of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells and skin tissue stem cells under the same conditions.
24. The method for evaluating a drug using human stem cells according to claim 23, wherein said step (2) further comprises embryonic stem cells.
25. The method of claim 24, wherein the same conditions comprise the same temperature, the same duration of action, the same concentration of the drug to be tested, the same contact pattern, the same ratio, the same generation of in vitro stem cells, the same oxygen concentration, the same carbon dioxide concentration, the same frequency of action, and the same number of repetitions.
26. The method for evaluating a drug using human stem cells according to claim 13, wherein in said step (2), the same test drug is allowed to act on the same stem cells in vitro under different conditions for the same stem cells.
27. The method for evaluating a drug using human stem cells according to claim 13, wherein in the step (3), the mortality and/or metabolic rate of the in vitro stem cells is detected; the effect of the drug on the stem cells is assessed by comparing the data before and after use of the drug or selected components of the drug.
28. The method for evaluating a drug using human stem cells according to claim 13, wherein the condition of the in vitro stem cells is detected a plurality of times in repeating said step (3) at different times.
29. The method for evaluating a drug using human stem cells according to claim 13, wherein in step (1), before starting step (2), the condition of the stem cells in vitro is detected; in the step (4), the detection result of the in-vitro stem cells in the step (1) is compared with the detection result in the step (3), and the influence of the drug to be detected on the in-vitro stem cells is evaluated.
30. A detection kit using human stem cells is characterized by comprising a box body, wherein the box body is filled with in-vitro stem cells; the in vitro stem cells are obtained by separating and amplifying in vitro organs and tissues of a human body; the in vitro stem cells comprise one or more of adult stem cells and/or embryonic stem cells.
31. The detection kit according to claim 30, wherein the adult stem cells include one or more of circulatory system stem cells, digestive system stem cells, respiratory system stem cells, nervous system stem cells, immune system stem cells, endocrine system stem cells, reproductive system stem cells, skeletal system stem cells, urinary system stem cells, muscle system stem cells, and skin tissue stem cells.
32. The test kit for detecting the presence of human stem cells as claimed in claim 31, wherein the stem cells comprise a plurality of types of stem cells, each of which is contained independently.
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