SIO2004 Animal Cell Culture Lecture 2

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Questions and Answers

What is the first step in initiating a cell culture?

Cutting tissue into small pieces

Adding media to the culture surface

Dissociating cells from the tissue

Taking a tissue biopsy (correct)

What method is used to dissociate cells from parental tissue for primary culture?

Thermal heating

Mechanical or enzymatic methods (correct)

Radiation exposure

Chemical synthesis

Which method does NOT help in isolating cells for culture?

Tissue biopsy

Dissociated cell culture

Cytotoxicity testing (correct)

Explant culture

Which statement accurately describes a primary culture?

It originates from dissociated parental tissue. Signup and view all the answers

In explant culture, where is the tissue fragment placed?

On a solid substrate at a liquid interface Signup and view all the answers

Which type of culture involves the continuous propagation of cells?

Cell line Signup and view all the answers

Which technique is primarily used for enzymatic dissociation of tissue?

Using trypsin or collagenase Signup and view all the answers

Which characteristic is true of primary cultures?

They contain a mix of adherent and non-adherent cells. Signup and view all the answers

What characterizes primary culture through explant culture?

Cells are migrated from a solid surface Signup and view all the answers

What is typically used as a container for growing dissociated cells in culture?

Glass or plastic containers Signup and view all the answers

Which of the following is NOT a method for dissociating tissue?

Applying heat to the tissue Signup and view all the answers

Which of the following best describes the nature of primary cultures?

They can be derived from both animal and plant tissues. Signup and view all the answers

Which statement about the cell migration in explant culture is correct?

Only adherent cells have the ability to migrate. Signup and view all the answers

What happens to the tissue fragments during the explant culture process?

They are placed intact to encourage cell outgrowth. Signup and view all the answers

What type of cells can be found in a 'tissue slurry' on a culture surface?

Both adherent and non-adherent cells Signup and view all the answers

What is the primary use of a primary cell culture derived from the lymphoid organ of Litopenaeus vannamei?

Study of WSSV replication Signup and view all the answers

What type of cells are first observed in a culture 24 hours after seeding?

Round cells Signup and view all the answers

Which cell type transforms into fusiform cells at 48 hours post seeding?

Round cells Signup and view all the answers

What is a key feature of a primary culture?

It is always heterogeneous. Signup and view all the answers

What happens to cell populations during repeated passaging of a primary culture?

Cells with high proliferative capacity dominate. Signup and view all the answers

Which approach is NOT a method for selecting a cell type of interest in a primary culture?

Applying excess nutrients Signup and view all the answers

What is the result of continuous passaging from a heterogeneous culture?

It eventually becomes homogeneous. Signup and view all the answers

After cell differentiation, which type of cells are observed at 72 hours post seeding?

Epithelioid cells Signup and view all the answers

What selective technique is mentioned for enhancing the recovery of desired cell types?

Flow cytometry Signup and view all the answers

What is the primary outcome of a primary culture undergoing several passages?

A homogeneous culture of the most vigorous cell type Signup and view all the answers

Which statement best describes the process of subculturing?

It is the transfer of cells to fresh vessels for continued growth Signup and view all the answers

What characterizes a continuous cell line?

It has undergone transformation to become 'immortal' Signup and view all the answers

What may happen to slow-growing cells in a mixed primary culture during passaging?

They may be lost due to competitive growth conditions Signup and view all the answers

How can specific cell types be selected in a primary culture?

By adjusting environmental conditions and using proper substrates Signup and view all the answers

What does a monolayer culture signify?

Cells are attached to a substrate and not in suspension Signup and view all the answers

What does cell sorting (flow cytometry) help with in culture techniques?

Selecting for specific cell populations Signup and view all the answers

What occurs when a cell line is established from a primary culture?

It will typically develop uniform characteristics over time Signup and view all the answers

What is a defining characteristic of Anchorage dependence in cell culture?

Cells require attachment to a substrate for growth. Signup and view all the answers

Which of the following cell types can survive and proliferate without being anchored to a substrate?

Hematopoietic cells Signup and view all the answers

What is a key advantage of using monolayer culture in cell studies?

It allows for easy visual inspection and quantification of cells. Signup and view all the answers

In suspension cell cultures, what factor is growth limited by?

Concentration of cells Signup and view all the answers

Which method is NOT typically used for dissociating adherent cells in culture?

Cryopreservation Signup and view all the answers

What is one application of suspension cultures?

Bulk protein production and batch harvesting Signup and view all the answers

What role do stem cells from Wharton’s Jelly play in COVID-19 treatment?

They increase the chance of survival in critically ill patients. Signup and view all the answers

What is one limitation of suspension cell culture compared to adherent cell culture?

Growth is limited by cell concentration. Signup and view all the answers

What distinguishes finite cell lines from continuous cell lines?

Finite cell lines die after several sub-cultures. Signup and view all the answers

Which cell type is categorized as spherical and typically grown in suspension?

Lymphoblast-like cells Signup and view all the answers

Fibroblast-like cells primarily migrate individually in low densities due to their requirement for what?

Substrate attachment Signup and view all the answers

Epithelial cells exhibit what kind of growth pattern due to their need for cell-cell adhesion?

Growth in patches Signup and view all the answers

What type of matrix protein do fibrocytes primarily secrete?

Type I collagen Signup and view all the answers

In terms of motility, which cell type is least motile?

Epithelial cells Signup and view all the answers

Which morphological characteristics correspond to fibroblastic cells?

Bipolar or multipolar with elongated shapes Signup and view all the answers

What enhances cell outgrowth in cultured tympanic membrane explants after physical trauma?

Physical trauma to the tissue Signup and view all the answers

Study Notes

SIO2004 Animal Cell and Tissue Culture, Lecture 2

Course: Biotechnology Program, University of Malaya
Instructor: Dr. Nuradilla Mohamad Fauzi
Topic: Cell Culture Initiation

Obtaining Cells for Culture

Cells are obtained via tissue biopsy.
Two primary methods for cell culture initiation: explant culture and dissociated cell culture.

Explant Culture

Tissue biopsy is taken.
Biopsy is dissected into small pieces.
Small tissue pieces (explants) are placed onto a culture surface.
Adherent cells migrate from explant.
Only cells capable of migration from the explant will develop in culture.

Primary Culture: Explant Culture

Tissue fragment placed on glass/plastic-liquid interface.
Cells attach, migrate in the substrate plane.
This process is observed in micrographs, showing tissue expansion and outgrowth over 24–48 hours.

Dissociation/Disaggregation

A tissue biopsy is taken.
Mechanical and/or enzymatic tissue dissociation.
Mechanical dissociation methods, e.g., serial pipetting, mincing tissue with a razor.
Enzymatic dissociation methods, e.g., trypsin and collagenase.
Tissue slurry is placed on the culture surface.
Both adherent and non-adherent cells survive dissociation.

Primary Culture: Dissociated Cell Culture

Maintenance of dissociated cells in culture medium.
Suitable glass or plastic containers used.
Dissociated cells form a monolayer at the solid-liquid interface.

Primary Explant Culture from Human Atrial Explants and C-Kit Positive Cardiac Stem Cells (CSCs)

Explant, initial outgrowth at 3, 7, and 14 days after explantation.
Confluent cell outgrowth away from explant after 28 days.
C-kit positive cells are sorted by flow-activated cell sorter.
A single, c-kit positive cell suspension is formed, resulting in a clone of human c-kit positive CSCs.

Selection of Cell Populations

Primary culture is heterogeneous.
Repeated passaging in standard culture conditions promotes proliferation of cells with high proliferative capacity.
Repeated passaging leads to a homogeneous final culture, which is a cell line.
Differences in growth rates in mixed primary cultures may result in loss of specific cell types.
Techniques used select for specific cell types include variations of medium, additives, substrates, and culture conditions.

From Primary Culture to Cell Line

Resulting monolayer/cell suspension is dispersed and subcultured into fresh vessels (subculture/passage).
Daughter cultures from subculture form the beginnings of a cell line.
After several passages, the culture becomes homogeneous.
Selective pressure of culture conditions leads to a homogeneous culture of the most robust cells.

Cell Line

Involves: (1) increase in total cell number over generations (population doublings); (2) predominance of cells, or lineages, with high proliferative capacity; and (3) uniformity in the cell population composition.
Cell lines retain specific characteristics throughout most of their lifespan.
Some cell lines can undergo transformation to become continuous cell lines (immortal).

Different Cell Types Behave Differently in Culture

Fibroblast-like cells tend to migrate individually.
Epithelial cells tend to grow in patches due to cell-cell adhesion requirements.
Some cells can produce their own extracellular matrix proteins (ECM) for attachment, such as type I collagen and fibronectin.
Other cells (e.g., stem cells) need components of the ECM added to the culture.

Morphology of Cells in Culture

Most mammalian cells can be divided into three basic categories based on morphology:
Fibroblastic (fibroblast-like cells): Bipolar or multipolar, elongated, attached to a substrate.
Epithelial-like cells: Polygonal, regular dimensions, attached in discrete patches to a substrate.
Lymphoblast-like cells: Spherical, grown in suspension without attaching to a substrate.
Cell morphology, partially corresponding to in vivo functions, can vary based on cell type

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Description

This quiz covers the key concepts of cell culture initiation discussed in Lecture 2 of the SIO2004 course. Topics include obtaining cells through tissue biopsy, the techniques of explant culture, and dissociation methods. Test your knowledge on the processes involved in establishing primary cultures.