MindMap Gallery Cell Culture and Practice
A mind map of cell culture and practice, which introduces the basis of cell culture, the application of cell culture in pharmacology and drug development, the application of cell culture in the field of immunology, stem cell culture technology and its application in regenerative medicine, cells Applications of culture techniques in pharmacology.
Edited at 2023-10-26 20:57:48This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
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This is a mind map about the reproductive development of animals, and its main contents include: insects, frogs, birds, sexual reproduction, and asexual reproduction. The summary is comprehensive and meticulous, suitable as review materials.
This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
This is a mind map about plant asexual reproduction, and its main contents include: concept, spore reproduction, vegetative reproduction, tissue culture, and buds. The summary is comprehensive and meticulous, suitable as review materials.
This is a mind map about the reproductive development of animals, and its main contents include: insects, frogs, birds, sexual reproduction, and asexual reproduction. The summary is comprehensive and meticulous, suitable as review materials.
Cell Culture and Practice
Basics of cell culture
1.Basic conditions for cell culture
cell culture laboratory
facility
equipment
2. Basic techniques of cell culture
(1) Aseptic technique
① Disinfection of operating range
wipe
UV irradiation
②Wash hands and dress
③Cleaning, disinfection and sterilization of culture supplies
Clean
Package
Disinfection and sterilization
④Aseptic culture operation
(2) Selection and processing of culture materials
①Source of embryonic and adult tissues
②Normal and tumor tissue sources
③How to obtain materials
(3) Cell culture types and technologies
① Primary culture
tissue block culture method
Preparation and culture of single cells
② Subculture
passage of tissue blocks
Subculture of adherent cells
Suspension cell subculture
(4) Cell morphology examination
① Observe cell morphology and structure with an inverted microscope
a. Ordinary staining observation—Giemsa staining, hematoxylin-eosin staining (HE staining)
b. Special staining method observation—special staining method observation, cell lipid staining, DNA/RNA staining
②Observe the living characteristics of living cells with a phase contrast microscope
Clearly visible light and dark contrasts between various structures within cells
③Use fluorescence microscope to observe the morphology and structure of cells
a.DNA/RNA acridine orange fluorescence staining method
b. Immunofluorescence staining method
④Use fluorescence microscope to observe the morphology and structure of cells
a.Transmission electron microscope (TEM)
b. Scanning electron microscope (SEM) - forming a three-dimensional structural image
(5) Detection and elimination of cultured cell contamination
① Microbial contamination and detection
a. Fungal contamination and detection—Aspergillus fumigatus, Aspergillus niger, Candida albicans, etc.
b. Bacterial contamination and detection—E. coli, Pseudomonas, Staphylococcus, etc.
c. Mycoplasma contamination and detection—the most common type of cell contamination
②Prevention and elimination of cell contamination
a. Preventive measures for cell contamination—establishing a cell bank, targeting sources of contamination, and targeting operations
b. Elimination of microbial contamination - antibiotic elimination method, heating sterilization, animal inoculation
c. Avoid cell cross-contamination
(6) Cell cryopreservation, recovery and transportation
Basic principles of cell cryopreservation and recovery: slow freezing and fast thawing
3. Cultured cell research and detection technology
(1) Cell cycle analysis method
①Cell cycle-S phase cells are most vulnerable to damage
②The growth process of cultured cells
③The life cycle of cells
a. Tissue cells in the body
b. Life cycle of cells cultured in vitro—short passage, limited passage, and immortality
④Cell cycle analysis
a. Flow cytometry: G0 phase, G1 phase, S phase, G2/M phase
b.Annexin V/PI double staining method: detecting cell apoptosis and cell membrane integrity
(2) Cell fusion
①Natural fusion
②Artificially induced fusion
③Homonuclear cell fusion
④Heteronaryotic cell fusion
(3) Cell cloning technology
The key to clonal culture is cell isolation
Measures to improve colony formation rate
Enhance cell assimilation capacity: such as fetal bovine serum, cytokines
Enhance the ability of cells to adhere to the wall: such as soft agar, preparation of bottom feeder layer cells
(4) Application of cell culture in virus propagation and vaccine production
Viral plaque; cytopathic effect
(5) Transformation of cultured cells
Infinite cell lines/continuous cell lines
(6) Apoptosis (programmed cell death)
①Morphological detection
②Biochemical testing
a.Agarose gel electrophoresis detection
b. Terminal deoxyribonucleotidyl transferase labeling detection (TUNEL)
c.Annexin V/PI detects cell apoptosis
d. Detection of hypodiploid DNA peaks to determine apoptosis
(7) Cultured cell gene injection technology
①Gene transduction technology
②Gene transduction technology
a. Chromosome transduction
b. Gene transfection
③Transgenic technology
(8) Cell enzyme activity measurement
Cytochemistry
a.Acid phosphatase
b. Glucose-6-phosphatase
c. Glucose-6-phosphatase
Cell culture applications in pharmacology and drug development
1. Effect of drugs on cultured cell proliferation
(1) MTT method
(2) Mitotic index method
2. Experiments on drug uptake by cells
3. Experiments on drug transport by cells
Application of cell culture in immunology
1. Preparation, isolation and purification of immune cells
Principles of immune cell isolation
①Based on the physical properties of cells
②Based on cell surface markers
(1) Preparation of peripheral blood mononuclear cell suspension
(2) Isolation and purification of lymphocytes
①Glassware adsorption method
②Magnet adsorption method
③Hydroxy iron-latex layered liquid method
④Adsorption column filtration method
(3) Separation of T cells and B cells
①Isolation of T cells and B cells by immunomagnetic bead method
② Use nylon wool column to separate T cells and B cells
③Separation of T cells and B cells by flow cytometry
(4) Isolation of T cell subsets
① Antibody/complement-mediated specific cytotoxicity to isolate T cell subsets (T subset)
②Panning separation method to isolate T cell subpopulations
(5) Isolation of monocytes and macrophages
(6) Isolation of monocytes and macrophages
2. Detection of the number of immune active cells
(1) Trace cytotoxicity test to detect the number of various types of immune cells
(2) Immunofluorescence staining technology to detect the number of various immune cells
(3) Flow cytometry to detect the number of various immune cells
(4) Immunoenzyme staining technology detects the number of various immune cells
3. Lymphocyte proliferation activity detection
(1) Principle of lymphocyte proliferation test
(2) Morphological examination of lymphocyte proliferation
(3) Morphological examination of lymphocyte proliferation by MTT assay
4. Detection of cytotoxic activity of immune cells
(1) Morphological examination method---Cytotoxicity test of lymphocytes against tumor cells
Stem cell culture technology and its application in regenerative medicine
1. Basic knowledge of stem cells
(1) Characteristics of stem cells
(2) Stem cell classification
(3) History of stem cell research
(4) Source of stem cells
(5) Differentiation potential of stem cells
(6) Differences between embryonic and adult stem cells
2. Embryo-derived pluripotent stem cells
(1) ES, EG, EC cells
(2) Isolation and culture of ES cells
(3) Self-renewal and differentiation capabilities of ES cells
3. Induced pluripotent stem cells
(1) Methods to reverse cell fate: dedifferentiation, reprogramming
①Nuclear transplantation
②Nuclear fusion
(2) “Pluripotency” transcription factors
(3) “Pluripotency” transcription factors
(4) Significance and problems of iPS cell research
4. Application of stem cells in regenerative medicine
(1) Concept of regenerative medicine
(2) Hematopoietic stem cell transplantation
①Classification
②Main indications
③Transplantation process
④Treatment steps and strategies
⑤Existing problems and research hot spots
⑥ Promotion of clinical applications
(3) Autologous limbal stem cell transplantation
(4) Application of neural stem cells
(5) Skin stem cells and beauty
①Basic structure of skin
②Two major advantages of autologous cell wrinkle removal
a. Effectiveness
b.Safety
(6) Other applications
①Myocardial repair
②ES cell therapy to treat diabetes
③Mesenchymal stem cells
(7) Relevant laws and regulations
Application of cell culture technology in pharmacology
1. Experimental cells
(1) Advantages and disadvantages of cells
(2) How to choose
Comparison of primary cells and cell lines/strains
(3) Research hot spots
cell growth cell differentiation
Cell proliferation and cell cycle regulation
Cellular aging and death
Cell surface receptors and signal transduction
vesicle transport
Mitochondria
Cytoskeleton
ion channel
Genome
2. Application in tumor research
(1) Experiment on affecting tumor cell growth (drug sensitivity test)
(2) Tumor cell migration test
(3) Tumor cell invasion test
(4) Establishment of tumor differentiation induction model
①Leukemia cell differentiation induction experiment
②Solid tumor differentiation induction model
(5) Detection of telomerase activity in tumor cells
(6) Detection of tumor cell genes and their expression products
①) Oncogene and tumor suppressor gene detection
②Oncogene and tumor suppressor gene detection methods
a. Radioimmunoassay method
b. Chemiluminescence immunoassay
c.Electrochemical immunoassay??
d. Immunocytochemistry technology
e.Flow cytometry
f.PCR technology
g. Genetic diagnostic technology
(7) Preparation and test of human tumor transplantation model in nude mice
3. Application in other pharmacology and pharmaceutical research and development
(1) Experiments on drug transport by cells (intestinal absorption)
(2) Constructing a human blood-brain barrier drug screening model
(3) Micronucleus test-toxicological application
cytometry
trypan blue exclusion method
Detection of cell viability and proliferation
Routine observation of cultured cells
Morphology, growth, culture medium color (pH), contamination or not
Viability detection of cultured cells
Trypan blue, MTT, CCK8, colony formation assay
Cell passage and maintenance
Change the medium, passage, cryopreservation and recovery.
Passaging method
A digestion method - adherent cells
B Direct pipetting method - some cells that are not firmly attached to the wall
C centrifugation or natural sedimentation method - suspended cells