Title: 134.231 Grundlagen der Zellkultur

Q: What is a "adherent cell culture"?
A: cells grow as monolayers attached to an artificial substrate

Q: What is "asepsis"?
A: means without infection or contaminating microorganisms

Q: What is "aliquoting"?
A: to separate a portion of the sample and place it in a separate tube

Q: What is "aseptic technique"?
A: Procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cell, tissue and organ culture. Although these procedures are used to prevent microbial contamination of cultures, they also prevent cross contamination of cell cultures as well.

Q: What is "cell culture"?
A: the maintenance of cultivation of cells in vitro including the culture of single cells. In cell cultures, the cells are no longer organized into tissues

Q: What is "cell generation time"?
A: The interval between consecutive divisions of a cell

Q: What is "Cell line"?
A: well characterized and (commercially) available type of cell. Cells with limited lifespan show characteristics of differentiation and can be propagated for only approx. 30 cell divisions; they senesce (stop multiplying). Other stem cell lines can be propagated infinitely due to high levels of telomerase

Q: What is "Cell strain"?
A: is derived from a primary culture or a cell line by the selection of cloning of cells having specific properties or markers. In describing a cell strain, its specific features must be defined

Q: What is "growth medium"?
A: A nutritive solution for culturing cells in which each component is specifiable and ideally, is of known chemical structure

Q: What is "confluency"?
A: refers to the percentage of the surface of a culture dish that is covered by adherent cells

Q: What is "Continuous cell culture"?
A: A culture which is apparently capable of an unlimited number of population doublings; often referred to as an immortal cell culture

Q: What is "cryopreservation"?
A: ultra-low temperature storage of cells, tissues, embryos or seeds. This storage is usually carried out using temperatures below -100°C

Q: What is "differentiated"?
A: Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo

Q: What is "electroporation"?
A: creation, by means of an electrical current, of transient pores in the plasmalemma usually for the purpose of introduction exogenous material, especially DNA, from the medium.

Q: What is "immortalization"?
A: The attainment by finite cell culture, whether by perturbation or intrinsically, of the attributes of a continuous cell line

Q: What is "Passage, split, subcultivate"?
A: the transfer of cells, with or without dilution, from one culture vessel to another

Q: What is "Passage number"?
A: the number of times the cells in the culture have been subcultured or passaged. In desciptions of this process, the ration or dilution of the cells should be stated so that the relative cultural age can be ascertained. some characteristics e.g. the sensitivity toward cellular stress may change with increasing passage number.

Q: What is "primary culture"?
A: a culture started from cells, tissues or organs taken directly from organisms. A primary culture may be regarded as such until it is successfully subcultured for the first time. It then becomes a "cell line"

Q: What is "Proliferation"?
A: cell proliferation is the process by which a cell grwos and divides to produce two daughter cells. Cell proliferation leads to an exponential increase in cell number. The total number of cells in a population is determined by the rate of cell proliferation minus the rate of cell death.

Q: What is "senescence"?
A: cessation of cell division. In vertebrate cell cultures, the property attributable to finite cell cultures; namely, their inability to grow beyond a finite number of population doublings

Q: What is "suspension culture"?
A: a type of culture in which cells, or aggregates of cells multiply, while suspended in liquid medium

Q: What is "transfection"?
A: the transfer, for the purposed genomic integration, of naked, foreign DNA into cells in culture
Non Viral!

Q: What is the difference between a primary cell culture and a cell line? How can you acquire them?
A: - Primary cell culture: Cells taken directly from tissues and cultured for the first time. They closely represent the tissue of origin and have a limited lifespan.
  - Cell line: Cells that have been subcultured multiple times and can proliferate indefinitely. They often acquire genetic mutations that allow continuous growth.
  - Acquisition: Primary cells can be obtained from tissue biopsy or surgery, while cell lines are typically acquired from cell banks like ATCC.

Q: Explain the term "subcultivation". What are the important steps in subcultivating adherent cells?
A: Subcultivation: Also known as passaging, it involves transfering cells from one culture vessel to antother to prevent overconfluence
Steps:
1. remove old media
2. rinse cells with PBS (phosphate buffer saline for maintaining pH, removing waste, minimize clumping of cells)
3. add trypsin to detach cells
4. incubate to facilitate detachment
5. neutralize trypsin with serum-containing media
6. transfer cells to a new vessel with fresh media

Q: You want to transiently express a flouroscent labeled protein in HeLa cells. How could you achieve this? Give a few examples of transient transfection methods. What are the advantages and disadvantages of transient transfection?
A: Achievement: lipofection, viral transduction, electroporation
Examples:
- Lipofection: use lipid-based reagents to deliver DNA into cells
- Electroporation: Use electric pulses to permeabilize the cell membrane
- Viral transduction: use viral vectors to deliver genetic material
Advantages: Quick, easy to perform, doesn't integrate into the genome (no mutagenesis)
Disadvantages: temporary expression (DNA degrades as it is not integrated) , potential for variable expression levels, and cytotoxicity

Q: Describe stable transduction and give examples +  advantages/disadvantages
A: Unlike transient transduction, here the foreign DNA gets integrated into the host cell's chromosomes, allowing the gene to be passed on to the daughter cells during cell divisions.
examples:
- viral vector (lentivirus, retrovirus) that get integrated into the host genome
- crispr integration
advantages:
- long term expression across multiple genereations of cells
- consistent expression levels: reliable results
disadvantages:
- complexity
- time consuming
- mutagenesis
hint: viral transduction can achieve both stable and transient transduction by utilizing different types of viral vectors and mechanisms.
e.g: lentiviruses -> integration into genome
adenoviruses -> short-term expression

Q: What is cell differentiation? Can this process be reversed?
A: Cell differentiation: The process by which a cell changes from one cell type to another, typically to a more specialized type.
Reversability: It can sometimes be reversed through a process called dedifferentiation, but is rare and usually occurs under specific conditions or in a lab setting with induces pluripotent stem cells (iPSCs)

Q: Which in vitro assays are performed to evaluate the cytocompatibility of materials?
A: MTT assay: Measures cell metabolic activity
Live/Dead assay: distinguishes live cells from dead cells using fluoroscent dyes
Alamar Blue assay: assesses cell viability through a colorimetric change
LDH assay: Measures lactate dehydrogenase released from damaged cells

Q: Describe thee zones found in multicellular spheroids. What does such an organization mimic?
A: Zones:
- Proliferation Zone: Outerlayer where cells actively divide
- Quiescent Zone: Middle layer with lower oxygen and nutrient levels, where cells are less active
- Nectrotic zone: Core where cells die due to lack of nutrients and oxygen
Mimic: This organization mimics the tumor microenvironment, with gradients of nuetrients, oxygen and waste products

Q: Which of the following is true?
- A: When freezing cells, they should be shock frozen to maintain cell viability
- B: Immortalized cells can be kept in active culture indefinitely
- C: Primary cells should be cultivated in phosphate buffer saline (PBS)
- D: the most commonly used buffer System for a cell culture medium is bicarbonate CO_2 buffer system
- E: A Class II Biosafety Cabinet is equipped witha HEPA Filter and offers protection of samples and the environment
- G: Porosity of a 3D scaffold material is important to maintain cell viability
A: A: true, minimizes formation of ice crystals
B: true, they are genetically modified to proliferate indefinately
C: false, should be grown in media, that has e.g.: nutrients, growth factors and hormones, not PBS
D: true, used to maintain the pH stability
E: false/true, BSL3 has HEPA filtration, BSL2 can have it in cabinets
G: true, porousity allows for better cell infiltration

Q: What equipment do you need for BSL2?
A: - controlled access
- hand washing sink
- sharp hazards warning policy
- physical containment device
- personal protective equipment (gloves, lab coat, goggles etc. )
- lab bench
- autoclave (for sterilization)
e.g.: HIV, HPV, COVID19

Q: What equipment do you need for BSL3?
A: - controlled access (double door, self closing)
- hand washing sink
- sharp hazards warning policy
- physical containment device
- personal protective equipment (gloves, lab coat, goggles etc. )
- lab bench
- autoclave (for sterilization)
- HEPA Filtration
- powered purifying respirator
- decontamination system

Q: What equipment do you need for BSL4?
A: - BSL3+
- protective suit (positive pressure)
- personal shower out
- chemical shower out

Q: What is a bioreactor? Why and how is it used in cell culture?
A: Bioreactor: A device or system that provides a controlled environment for the growth of cells or tissues
Use: It supports large-scale cell culture, provides controlled conditions (temperarue, pH, oxygen) and allows for high-density cell growth
How: Cells are cultured in the bioreactor with continuous monitoring and adjustments of environmental parameters.

Q: What is a tripotential differentiation assay? What are the steps involved?
A: Assesses the ability of stem cells to differentiate into three germ layers:
- ectoderm (skin, neural tissues, sensory organs)
- mesoderm (muskle, bone, blood, connective tissue)
- endoderm (lining of digestive/respiratory tissues)
Steps:
- Induction (Cultivation of stem cells in special media)
- Differentiation (Allows cells to differentiate over time)
- Analysis (Evaluate differentiated cells using specific markers)

Q: Which of the following statements are true?
A: Cell differentition in reverse is not possible
B: Most used buffer system: Bicarbonate/CO_2 buffer
C: Cell line is subcultured at 37°C in laminar flow
D: Cryoprotectant: 1°C/min.
A: A: false, it can be reversed under certain, but rare conditions
B: true -> maintain certain pH level
C: true for temperature, laminar flow is not neccessary, unless sterile conditions are required
D: true, a controlled cooling rate is essential for cryopreservation

Q: What aspects should be taken into account if you want to do in vitro metabolic assays?
A: - Cell type: Choose the appropriate cell type for the assay
- Assay conditions: Ensure optimal conditions (pH, temperature)
- Reagents: usage of high-quality reagents
- Time points: collect data at multiple time points to understand metabolic changes over time

Q: What are the main advantages of 3D cell cultures?
A: - mimics the natural environment of cells
- better cell-cell and cell-matrix interactions
- Improved drug testing due to more accurate prediction of in vivo responses
- Study of tissue archtecture (specific functions and structures)

Q: True or False?
A: Cells should be cooled as fast as possible
B: Stem cells cannot be proliferated indefinitely
C: BSL2 protects probe and environment and has HEPA filters
D: most used buffer system: bicarbonate/CO_2 Buffer
A: A: false, cells should be cooled at a controlled rate to prevent damage
B: false, under the right conditions, they can proliferate indefinitely
C: true, but HEPA filters at BSL2 have different task compared to ones ins BSL3
D: true, bc pH control

Q: What is the difference between primary cell cultures and cell lines? Where can you get them from?
A: Difference: primary cells are directly taken from tissues and have a limited lifespan, while cell lines can proliferate indefinitely due to mutations
Sources: Primary cells can be obtained from tissue biopsies or surgeries, cell lines can be acquired from cell banks such as ATCC

Q: What kind of vessels are used for subcultivating adherent cell lines? Why is it important?
A: Vessels: Typically flasks, petri dishes, or multi-well plates
Importance: Ensures cells have enough space and nutrients to grow without becoming overconfluent, which can affect their physiology

Q: Describe the main steps for subcultivating adherent cell lines
A: 1. remove old media
2. rinse cells with PBS
3. add trypsin to detach cells
4. incubate to facilitate detachment
5. Neutralize trypsin with serum-containing media
6. transfer cells to a new vessel with fresh media

Q: What procedure would you suggest using if you want HeLa cells to transiently express a fluorescently laberled protein? What methods are there to transiently transfect cells? What are the advantages and disadvantages?
A: Procedure:
Use a transient transfection method such as lipofection, electroporation or viral transduction
Advantages:
Easy and fast, doesn't integrate into the genome
Disadvantages:
only temporary expression, variable levels of expression, potential cytotoxicity

Q: What is cell differentiation? Can it be reversed?
A: Differentiation: Process by which cells become specialized
Reversibility: Possible through dedifferentiation, typically in a lab setting with iPSCs

Q: What are the characteristics of adherent cells compared to suspension cells?
A: Adherent cells: grow attached to a surface, require specific substrates. They form a monolayer on the surface and require a trypsinization for passaging
Suspension cells: grow freely in the culture medium, don't need attachment, are easier to subculture as they can be simply split by transferring a portion of the culture to a new vessel

Q: How can you track fluorescent proteins in cells?
A: Using flourescence microscopy of flow cytometry

Q: How is protein staining performed in cell biology?
A: Involves fixing the cells, permeabilizing the membrane and using specific antibodies conjugated to fluorescent dyes (immunofluorescence) or chromogenic substrates (immunohistochemistry) to visualize the targe protein under a microscope

Q: What is needed for BSL1?
A: - controlled access
- hand washing sink
- sharp hazards warning policy
- personal protective equipment
- laboratory bench
- (optional depending on hazard) autoclave

Q: Which phases of proliferation do you know?
A: 1. Lag phase: acclimation of cells, they do not divide during this phase
2. Log (logarithmic) phase: actively dividing during this phase, best time for assessing population growth. Late in this phase is the best time to passage
3. plateau (stationary phase): cell growth in this phase slows as cells approach 100% confluence
4. Decline Phase: cell death predominates in this phase

Q: In which parts can the cell cycle be divided?
A: G1-Phase: protein syntheses, growth
S-Phase: DNA-replication
G2-Phase: synthesizes enzymes that control cell division, check of DNA replication and error repair
M-Phase: mitotic phase, cell replicates its nucleus and then pinches in two to form two new daughter cells
G0-Phase: Cells "rest"

Q: What is the Hayflick limit?
A: the number of times a normal human cell population will divide before cell division stops
Each time the telomeres shorten until it can't no more divide

Q: How to make immortalized cells?
A: - induces telomerase
- induce/introduce oncogenes (cancer) that will allow cell cycle to continue

Q: What is the difference between transfection and transduction?
A: transduction is concerned with viral methods
transfection is NOT viral

Q: Describe transfection and give examples +  advantages/disadvantages
A: stable transfection -> integration into genome
transient transfection -> no integration into genome
examples:
- electroporation (transient)
- chemical transfection with cationic lipids(transient)
- microinjection (mostly transient)
Advantages:
- less mutagenesis due to mostly only transient transfection
Disadvantages:
- expression levels can vary
- due to mostly transient transfection also only temporary expression

What is immunofluorescence and how can it be achieved?