Zellkulturen

• cells grow as monolayers

• attached to artificail substrate

without infecting, contaminating microorganisms

• used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cell, tissue and organ culture

• sterile working area

• good personal hygiene

• sterile reagents & media

• sterile glass & plasticware

• to separate a portion of sample

• and place it in different tube

• maintanance / cultivation of cells in vitro including culture of single cells

• in cell culture: cells are no longer organized into tissue

• interval between consecutive divisions of a cell

= derived from a primary culture after the cells undergo several rounds of division. They can become immortalized either spontaneously or by genetic manipulatio

• well characterized

• (commercially) available type of cell

• limited lifespan = characteristics of differentiation

• only max. 30 cell divisions

• senesce (grow old)

• population of cells that arise from a cell line with a distinguishing feature

• Obtained through positive selection of cells, which have undergone a particular biotechnological approach such as cloning

• have additional genetic change other than the cells of its parent cell line responsible for its distinguishing feature.

• nutritive solution for culturing cells

• each component = specifiable & ideally & on known chemical structure

• refers to percentage of surface of culture dish

• that is adherent cells covered

• culture which is appearently capable of unlimited number of population doublings

• “immortal cell culture”

• ultra low temperature storage of cell, tissues, embryos, seeds

• temp below -100°C

• process by which a cell becomes specialized to perform a specific function

• cells that maintain (in cell culture) all / much of specialised structure

• function typical of cell type in vivo

• creation, by means of an electrical current, of transient pores in plasmalemma

• for purpose of introducing exogenous material = DNA from medium

= Methode, Zellmembranen vorübergehend permeabel (durchlässig) zu machen, um so Makromoleküle, wie DNA oder Proteine, in Zellen oder Gewebe einzuschleusen

• attainment by finite cell culture = perturbation / intrinsically

• of attributes of continous cell line

• transfer of cells

• with / without dilution

• from one vessel to another

• number of times the cells in culture have been subcultured / passaged

• rotation or delution of cells should be stated = sp relative cultural age can be ascertained

• some characteristics zB. sensitivity towards cultural stress may change with increasing passage number

• culture derived directly from tissue or organ samples of an organism and is cultured for the first time.

• may be regarded as such UNTIL it’s subcultured for first time

• then becomes “cell line”

• process by which a cell grows & divides to produce 2 daughter cells

• leads to exponential increase in cell number

• total number of cell = rate of cell proliferation MINUS rate of cell death

• cessation of cell division

• in vertrebrate cell culture: property of attributes to finite cell cultures = inability to grow beyond finite number of population doublings

• type of culture in which cells / aggregates of cells multiply while suspended in liquid medium

• maintanance of growth of tissues, in vitro

• in a way that may allow differentiation & preservation of their architecture / function

= growth of tissues or cells in an artificial medium separate from the parent organism “micropropagation”

• process of artifically introducing nucleic acids (DNA, RNA) into cells

• to study gene function & protein expression

• 1. Chemical transfection: complex formation (pos, neg)

  2. physical transfection: microinjection, electroporation

• involves transferring cells from one culture vessel to another ro provide fresh nutrients & space for continued growth

• Vessels: flasks, petri dishes, multi-well plates

• Importance:

  • prevents over confluency

  • maintains healty cell growth

  • essential for long term cell culture maintanece

• Steps: Detach cells using trypsin or EDTA, count cells, dilute and replate in new vessels

Method: use molecular biology techniques to transfer cells with plasmids encoding fluorescent protein (GFP, RFP)

• lipofection

• electroporation

• viral transduction

expression & localization of fluorescent protein can then be observed using fluorescence microscopy

• by isolating cells from tissue

• through enzymatic (trypsin, collagenase) / mechanical means

• then plating them into suitable culture medium

• require surface to attach & grow

• require specific substances = collagen & fibronectin

• spread out on culture surface - form monolayer - show specific cell morphology

Def: method to introduce nucleic acids into cells temporarily

Methods: Lipofection, electroporation, viral transduction

Advantages: rapid expression of gene of interest, do not require stable integration into genome + useful for short term studies

Disadvantage: transient expression, variability between cells, lower efficiency in some cell types

• require attachment to substrate for growth

• examples include fibroblasts & epithelial cells

• grow freely in culture medium

• example include blood cells & certain cancer cell lines

• cell lines that proliferate indefinetely due to genetic modifications / naturally occuring mutations

• zB: HeLa cella (derived from cancer cells)

• process by which cells become specialized

• gold standart for cytotoxicity testing to measure cellular metabolic acitivity

• used to assess cell viability & proliferation

• zB: MTT assay, XTT assay, resazurin-based assay

Vorteil: simple, high throughput

Nachteil: low sensitivity, toxicity, chemical interference

1. Proliferative Zone: The outermost layer where cells have ample access to nutrients and oxygen. Cells in this zone are actively dividing and proliferating.

2. Quiescent Zone: Located just beneath the proliferative zone, this layer has limited nutrient and oxygen availability. Cells here are generally in a dormant or quiescent state, not actively dividing but still viable.

3. Necrotic Zone: The core of the spheroid where nutrient and oxygen levels are critically low or non-existent, leading to cell death and necrosis

• mimics the microenvironment of solid tumors found in vivo

• used to visualize & quantify proteins in gels (SDS-PAGE)

• common stains include Coomassive Brillant Blue & Silver stain

• process by which cells develop in reverse (reversed differentiation)

• from more differentiated into less / stem cell like state

• observable at genetic level of:

  • gene expression

  • proteins

  • morphology

  • function

• unwanted in cell culture, present in amphibians

• Class II Biosafety Cabinet (BSC): Provides both sample and environmental protection through HEPA filtration.

• Autoclave: For sterilizing equipment and disposing of biohazardous waste.

• Centrifuge with sealed rotor: To safely handle and process potentially infectious samples.

• Personal Protective Equipment (PPE): Including lab coats, gloves, and safety goggles.

• Handwashing Sink: With soap and disinfectants.

• Eyewash Station and Safety Shower: In case of exposure to hazardous materials.

• Proper Waste Disposal Containers: For sharps, biohazardous waste, and chemical waste.

• 3D cell aggregated that mimic the in vivo environment better than 2D cultures

• used in cancer research, drug testing, tissue engineering

1. Plasmid Construction:

   • Clone the gene encoding the fluorescent protein (GFP, RFP) into a suitable expression vector with a strong promoter (CMV promoter)

2. Transfection:

   • Transfect HeLa cells with the plasmid using a transfection reagent (e.g., Lipofectamine, PEI) or electroporation

3. Selection:

   • Include a selectable marker gene in the plasmid (antibiotic resistance gene such as neomycin or puromycin resistance)

   • After transfection: treat cells with the appropriate antibiotic to select for stably transfected cells. Only cells that have integrated the plasmid into their genome will survive

4. Cloning:

   • Perform limiting dilution or single-cell sorting (flow cytometry) to isolate individual clones

   • Expand individual clones and screen for fluorescence using a fluorescence microscope or flow cytometer

5. Verification:

   • Confirm stable integration and expression of the fluorescent protein by PCR, Western blot, or fluorescence imaging

= device or system used to grow cells or tissues in controlled conditions. It is especially useful for large-scale cell culture and tissue engineering

Why:

• Scalability: Allows for the cultivation of large volumes of cells, producing greater yields than standard culture methods.

• Control: Provides precise control over environmental conditions such as pH, temperature, oxygen, and nutrient supply.

• Homogeneity: Ensures uniform exposure of cells to nutrients and gases, leading to more consistent cell growth and function.

How:

1. Inoculation: Seed the bioreactor with an initial population of cells.

2. Monitoring and Control: Continuously monitor and adjust the culture conditions using sensors and control systems to maintain optimal growth conditions.

3. Harvesting: Once the cells have grown to the desired density, harvest them for downstream applications.

• used to assess the differentiation potential of stem cells into three germ layers: ectoderm, mesoderm, and endoderm

Steps:

1. Stem Cell Culture:

   • Culture the pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) under undifferentiated conditions.

2. Differentiation Induction:

   • Induce differentiation using specific media or growth factors for each lineage:

     • Ectoderm: Typically involves factors like retinoic acid.

     • Mesoderm: Often induced with factors like BMP4.

     • Endoderm: Usually involves factors like Activin A.

3. Differentiation Confirmation:

   • Confirm differentiation using lineage-specific markers through techniques such as immunostaining, qPCR, or flow cytometry.

   • Ectoderm markers: Nestin, Sox1.

   • Mesoderm markers: Brachyury, Desmin.

   • Endoderm markers: Sox17, AFP.

4. Functional Assays:

   • Perform functional assays to verify the differentiated cell types, such as electrophysiological tests for neurons (ectoderm), contractility assays for muscle cells (mesoderm), and secretion assays for liver cells (endoderm).

Die drei Keimblätter sind Gewebecluster, die sich im Rahmen der Embryogenese bilden. Sie entstehen nach der Befruchtung aus der Zygote durch Zellteilung

• inorganic salts - osmotic balance, regulate membrane potential (sodium, potassium, calcium inons)

• Glucose - main carbon source (what they eat), pyruvic / latic acids (1-4.5g/L) & CO2

• L-glutamine - additional carbon source

• Serum of animal blood - (bovine / equin) - growth factors & hormones

• vitamins - co factors, VitB groups for cell growth & proliferation, sometimes B12

• phenol red - pH indicatior, pH 7.4 (requires app. buffer system = carbonate)

• antibiotics - penicilin, streptomicin = against gram positive / negative bacteria

• buffer system - bicarbonate / CO2 buffer, (HEPES)

correct & stable growth conditions

• T = 37°C

• CO2 = pH regulation in bicarbonate, CO2 buffering system 5%

• Humidity = gas exchange with the atmosphere = evaporation of media must be prevented = humidity 100%

1. Bacteria

2. Mycoplasm

3. Fungi

4. Viruses

• for work involving well-characterized agents NOT known to consistently cause disease in immunocompetent adult humans

• present minimal potential hazard to laboratory personnel and the environment

1. Mechanical pipetting only (no mouth pipetting allowed)

2. Safe sharps handling

3. Avoidance of splashes or aerosols

4. Daily decontamination of all work surfaces when work is complete

5. Hand washing

6. Prohibition of food, drink and smoking materials in lab setting

7. Personal protective equipment, such as; eye protection, gloves and a lab coat or gown

8. Biohazard signs

• for work involving agents of moderate potential hazard to personnel and the environment

• Laboratory personnel: specific training in handling pathogenic agents and are directed by scientists with advanced training

all from BSL 1 plus:

1. Appropriate personal protective equipment must be worn + lab coats and gloves + Eye protection and face shields can also be worn

2. All procedures that can cause infection from aerosols or splashes are performed within a biological safety cabinet

3. An autoclave or an alternative method of decontamination is available for proper disposals

4. The laboratory has self-closing, lockable doors

5. A sink and eyewash station should be readily available

1. Lag phase: cell attachment within 24h after initiation of culture length determined by growth phase & seeding density

2. Log phase: cells are activ, best time for assessing population growth + data collection

3. Plateau phase: cells grow slowly as they approach 100% confluence

4. Decline phase: neutral part of cycle = cell death

• number of times a normal human cell population will divide before division stops

• by inducing:

  • telomerase

  • oncogenes (allow cell cycle to continue)

• first stable cell line isolated by Gregor gex (1951)

• from cervix adenocarcinome

• used to test frist polio vaccines & first successful cloned human cells

• Fibroplastic cells: murine embryontic fibroblasts 3T3

• Epithelial like cells: human cervix cells

• Lymphoblast like cells: blood cells

• contamination being established

• although parts of supposed cell lines are still genuine

  
  

• during establishment of original cell line

• some contaminating cells accidently introduced into cell cultures & intime outgrow desired cells

• sample / portion of total amount of solution

• G1: protein synthesis + growth

• S: DNA replication

• G2: enzymes for controll of cell division + check of DNA + error / repair

• M: Mitotic phase = 2 new daughter cells

fluorescence in situ hybridisation

• fluorescent probes that bind only to parts of chromosomes with high degree of sequence complimentarity

• zB: gene mapping on chromosomes, mapping expression cells

• multicolor fish technique

• 5 different fluor. = combi allows identification of all 24 human chromosomes

• spectral image analysis

• short tandem repeats of 2-7 bp

• distributed through gene

• variieren in Anzahl an Wiederholungen = ideale Merkmale für Identifikation

• zB: Vaterschaftstest, forensische Untersuchungen, Populationsgenetik

• to measure expression levels of large numbers of genes

  1. extract mRNA

  2. make & laben DNA

  3. hybridize to spotted oligos

  4. scan & analyze

• cells imaging technique

• uses antibodies to detect specific target antigen

  • specifically binds to target region on antigen

  • part of immunesystem (produced by mammals)

• Direct labelling: with fluorescently labelled antibody that recognizes protein of interest

• Indirect labelling: with fluorescently labelled antibody that recognizes primary antibody

• laser based technique to detect & analyze cells

• cells supended in stream of fluid & passed through electronic detection apparatus

fluorescence activated cell sorting

• allows sorting a heterogenous mixture of biological cells into 2 or more containers

1. sdf

2. safds

3. asdfs

4. sadfs

• differantiate from normal / non-specialized cells to specialized tissue cells

  
  

• most versatile (vielseitig)

• able to differentiate into any kind of cell

  
  

• little limited differentiation range

• can only develop into 2 different cell types

• addition of human growth hormones

• change culture conditions in right time

• monitor differentiation via specific markers

number of healthy cells in sample

Parameters:

• redox potential

• integrity of cell membrane

• activity of cellular enzymes

• natural process

• cells dying & being replaced by new ones

• non physiological process

• result of infection / injury

1. Quiescent cell:

   • performing its function without actively preparing to divide

2. Sesecent cell:

   • permanent cell growth arrest in normal & altered physiological processes

• living cells with intact membrane = will exclude dye

• dead cells = will uptake dye due to impaired membrane integrity

Vorteil: cheap & straight forward & quick

Nachteil: low sensitivity & endpoint assays

1. Calcein AM: viable cells = green

2. Intercalating dye: dead cells = red

• blue fluorescent DNA stain

• excitate 405 nm

• binds to dsDNA minor groove at A-T rich regions

• “nuclear counterstain”

VS. Hoechst dye: less toxic = higher cell viability, more cell membrane permeable

Fluorescent dye binding to dsDNA

• balance between energetic demands & biosynthetic requirements to support cellular function

• met. functions:

  • generate energy

  • building blocks for protein

  • elimination of metab. waste

  (Metochondria)

• 
  

• reducing power of living cells to convert:

• resazurin to fluorescent resofurin

• by reduction of: NADH, NADPH, FADH

Vorteil: ready to use, low cost, quick, sensitive, for 2 & 3D

• Determining the number of viable cells in culture based on quantitation of the ATP

• in vitro test that determine bio. reactivity of mammalian cell cultures to contact with material

Methods:

number of cells (DNA Quantification)

proliferation, viability (Live/Dead viability assay or Trypan Blue)

metabolic activity (MTT, PrestoBlue, CellTiter-Glow)

Morphology of cells (microscope

• Alizarin red S – the gold standard for quantification of osteoblast mineralization

• anionic dye binds to cationic metals such as calcium

• stain reacts with calcium cation & forms bright red chelate end product

• used to visualize acidic epithelial and connective tissue mucins

• Used to identify chondrocytes from differentiated cultures of mesenchymal stem cells

• cationic dyes: bind strongly to sulfated GAGs and glycoproteins

  
  

• foreign DNA integrated into genome = permantent genetic alteration

• selection for 2-3 wochen

• lower protein expression

• inducible system possible

• foreign DNA does not integrat into genome = cells lose plasmind

• use cells after 24-48hr after transfection (short term expression)

• high protein expression

• inducible system impossible

= uses viral vectores to deliver DNA / RNA to cells

1. Retro/Lentiviral: foreign DNA is integrated into genome (BSL 2) : RNA-viruses

2. Adenovirus -> transient transduction: foreign DNA is not integrated into genome DNA virus