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Kevin Suzuki

TE 2015
1) What are some tissue types that we might want to replace?
Epithelial and Connective Tissues , damaged tissues, infected tissues, etc.
2) What do autologous, allogenic, syngeneic, and xenogenic mean?
Autologous: Donor to him/herself, Allogenic: Donor to recipient of same species,
Syngeneic: Genetically identical donor (homozygous twin), Xenogenic: Cross species
(animal to human)
3) What is a cell-based therapy?
Cell based therapy is a therapy that requires cells to be used as therapeudic agents
to affect a harmful condition.
4) What is the origin/history of tissue culture? What is the difference between tissue
culture and cell culture in practice?
Tissue culture began in the beginning of the 20 th century as a method for studying
the behavior of animal cells free of systemic variations that might arise in vivo both
during normal homeostasis and under the stress of an experiment. It originally
began with small fragments of tissue and the growth was limited to the tissue
fragments. Tissue culture is a generic term which includes organ culture and cell
culture, whereas cell culture is a culture derived from dispersed cells taken from
original tissue.
5) What are some advantages and disadvantages of tissue culture (or in vitro
experiments in general)?
Two main advantages are the fact that physiochemical environmental factors like
pH, temperature, O2, etc can be controlled precisely and the physiological
conditions can be kept relatively consistent with specific experimental compounds
being regulated in specific concentrations. However, some of the downsides are the
fact that these techniques have to be kept in sterile conditions and there needs to
be a level of skill and understanding, there is a major limitation in quantity and cost
of producing cells which can be high when trying to prepare a large sample, there is
also dedifferentiation which can make it difficult to find the original tissue samples
from which these cultured cells are derived, and there is also instability which can
cause major problems.
6) How does a biological safety cabinet (Class II) work?
The Class II safety cabinet works by primarily using a recirculating fan which then
allows clean air inside the cabinet and then leads to the work area. Then the air
goes underneath the work area and is passed through the top of the cabinet and

leaves but cant come back because of the pathogen trap filter and the flap valves
to prevent blow back.
7) What are autocrine, paracrine, and endocrine signaling?
Autocrine: cell signals itself through a chemical that it synthesizes and then
responds to solely within the cytoplasm of the cell or by a secreted chemical
interacting with receptors on the surface of the same cell.
Paracrine: chemical signals that diffuse into the area and intact with receptors on
nearby cells.
Endocrine: chemicals are secreted into the blood and carried by blood and tissue
fluids to other cells
8) Describe, semi-quantitatively, what happens to the cell number after primary
culture. What are the differences between normal and transformed cells?
After primary culture, the cell number increases because they become more
confluent and spread until the surface of the dish is all covered. Transformed cells
are cells that have exogenous genetic material within itself. The main difference is
the changed functionality, DNA configuration, etc.
9) What are the roles of the extracellular matrix?
The ECM provides support, segregates tissues from one another and regulates
intercellular communication. It is also composed of proteins and sugars
10) What are mesenchymal cells? Epithelial cells? What are some of their defining
characteristics? What tissues do they come from?
Mesenchymal cells exist alone as small or loosely connected cells that are able to
develop into tissues in the body as well as connective tissues like bone and
cartilage. They are derived from the mesoderm and differentiate into hematopoietic
stem cells. Epithelial cells are 2d sheets which are tightly connected and cannot
individually migrate. Epithelial cells are found in major cavities of the body and form
skin, lungs, cell lines and specialize to act as sensory receptors. Epithelial cells form
boundary and protection, sensory sensations, transportation, absorption, secretion
and lubrication and movement. Epithelial cells derive from cells that line the inner
cavities of the body.
11) What are some of the relevant length scales in tissue engineering? Do you also
find the hospital picture a bit cheesy? Do you buy their length scales or are there
others that might be important?
1:100, and 1:200 are two scales. And Im sure there are other scales that may be
important but I do not think that we care about super specificity on the scales.

12) What are some common cell culture substrates? Why might you choose one
over another?
Glass and disposable plastic are two types of common cell substrates. Glass is good
because it is easily washed without losing its growth-supporting properties and can
be sterilized readily by dry or moist heat and is optically clear. Disposable plastic is
good because they are of good optical quality and the growth surface is flat,
allowing for a uniformly distributed and reproducible monolayer culture. Polystyrene
is the most common and cheapest.
13) What is the purpose of keeping cells in the incubator?
The purpose of keeping cells in an incubator is to provide cells with the optimum
temperature, moisture, and optimal pH for them to grow and maintain life.

14) What role does CO2 play? (Use as much chemistry as you can).
CO2 dissolves the medium and establishes equilibrium with HCO3- and lowers the
pH. The atmospheric CO2 tension regulates the concentration of dissolved CO2
directly as a function of temperature.
H20+CO2<-> H2CO3<->H+ + HCO315) Why is there phenol red in many media?
Phenol red is used as an indicator because it is red at pH 7.4, orange at 7.0, yellow
at 6.5, super yellow below 6.5 and super purple above 7.8
16) What is the importance of salt in media? Glucose?
A balanced salt solution is comprised of inorganic salts and can include sodium
bicarbonate and glucose. Salts contribute to the osmolality of the medium and
glucose is included as a source of energy.
17) What is serum? What does it do? What are the advantages and disadvantages
of using it?
Serum contains growth factors and promotes cell proliferation, and adhesion factors
and antitrypsin activity which promote cell attachments. It is also a source of
minerals, lipids and hormones which bound to proteins. We use FBS (fetal bovine
serum). The disadvantage of using serum is that it can be expensive, have problems
with standardization, specificity or could have unwanted effects like stimulation or
inhibition/growth, etc.
18) What is a hemocytometer?

A hemocytometer is a plate used for counting cells and is organized into 9 equal
sized grids where you count the 4 corners and the center square and average the
number of cells to get your average number of cells.
19) What are primary cells? What are their advantages and disadvantages?
Primary cells are cells taken directly from living tissue and established for growth in
vitro. Advantage: primary cell lines grow faster and need minimal care.
Disadvantage: there is a possibility that cell lines may behave differently in
comparison to primary cells in response to stress.
20) Where might we get primary cells experimentally? For clinical use? Advantages
and disadvantages of various sources??
Primary cells can be taken from mice and chickens?? Is this what the question is
asking?
21) What are some general approaches to isolating primary cells?
To isolate primary cells, there are purely mechanical techniques involving dissection
with or without some form of maceration and techniques utilizing enzymatic
disaggregation. Specifically, fine dissection includes primary explants, mechanical
disaggregation involves sieving, syringing or vigorous pipetting and enzymatic
disaggregation involves cold trypsin, warm trypsin and collagenase. (p183)
22) What are collagenase and trypsin and why might they be useful?
Collagenases are enzymes/bacterial proteases which are not neutralized by trypsin
inhibitors. Trypsin is a serine protease found in digestive system of many
vertebrates. They are useful for tissue disaggregation to remove cells off the dish.
23) What is subculture? Confluence?
A subculture is a process by which the tissue is first subdivided and then transferred
into fresh culture medium. Confluence is when the surface of the plate is covered by
cells.
24) What is a cell line (NOTE: there is some discrepancy in the older definition and
how this word is actually used)?
A cell line is a cell culture developed from a single cell and consists of cells with
similar or distinct phenotypes. Once a primary culture is subcultured (passed), it
becomes a cell line.
25) Why do we change the media on cells? Why do we subculture them (a.k.a.
passage them)?

The media on the cells are changed in order to keep the cells healthy by providing
fresh nutrients. Cells are subcultured in order to allow them to grow more and allow
scientists to keep cells alive under cultured conditions for extended periods of time.
26) What is contamination and what are some forms it comes in?
Contamination is a substance or a malpractice which can have a significant impact
on the final outcome of the experiment and can come in forms in chemical
contaminants like impurities in media, sera and water, and biological contaminants
like bacteria, molds, yeast, etc.
27) What are some sources of contamination? What are some steps we take to
avoid contamination?
Sources of contamination can arise from failure in the sterilization procedure for
solution, glassware and pipettes, turbulence and particulates (dust and spores),in
the air in the room, poorly maintained incubators and refrigerators, faulty laminarflow hoods, the importation of contaminated cell lines or biopsies, and lapses in
sterile techniques. In order to avoid contamination, you can clear work area of items
not in immediate use, wash hands and wear gloves, sterilize items by dry heating
them, use clean filtered air, throw away all things if not being reused, etc.
28) Why might we want to cryopreserve (freeze) cells? Engineered tissues?
Cryopreservation of cells is essential because the use of each cell line adds to its
provenance and if they are unique, each one becomes a valuable resource and
impossible to replace; thus it is important to reserve them to protect the cells.
Specifically, cells are frozen to prevent genotypic drift due to genetic instability,
contamination by microorganisms, need for distribution to other users, incubation
failure, etc.
29) What are the properties of stem cells? Where are they found?
Stem cells are capable of dividing and renewing themselves for long periods; they
are unspecialized; and can give rise to specialized cell types. Adult stem cells are
found in bone marrow or fat cells, whereas embryonic stem cells come from
embryos.
30) What is a pluripotent cell? Progenitor cell?
Pluripotent cells are true stem cells because they have the potential to
differentiate into almost any cell in the body. A progenitor cell is a cell that is more
specialized than the pluripotent cells, but still can be pushed to differentiate into its
target cell.
31) What are differentiation and de-differentiation? How can we detect them?

Differentiation is the phenotypic change to specialized cell types and


dedifferentiation is the process where partially or terminally differentiated cells
revert to an earlier developmental stage due to a regenerative process. We can
detect differentiation and dedifferentiation by using DNA chips or microarrays.
32) What are some ways we can induce differentiation?
Differentiation can be induced
33) What are the 3 dynamic states of tissues (according to Palsson & Bhatia)?
Tissue homeostasis: normal-steady function of tissue, Tissue Repair: Display a
healing response, Tissue formation: formation of tissue
34) What is morphogenesis? What are some of the processes that are involved?
Morphogenesis is the evolution and development of form. It involves coordinated
activity of many cells and most tissues want to begin development until cells of
different type come into physical contact with one another. The steps involved are
Initiation: Where a starting signal destabilizes intracellular arrangement and drive
cells to initiate activities, After initiation: Spatio-temporal process which is a robust
convergence of the final fate and its Final State: No more morphogenic driving force
and stabilization.
35) What are receptors and ligands and what are their roles? What are some
examples?
Receptors and ligands are protein molecules which are embedded in the plasma
membrane of a cell that receives chemical signals from the outside of the cell and
leads to some form of cellular/tissue response. Examples include integrins,
cadherins, collagen, fibronectin, bromide, sulfide, fluoride, water, etc.
36) What are some ways that we might analyze cells in regular culture (2D)?
Morphology, genetics, density, motility, microscopy, etc , are ways to analyze cells
in 2D.
37) Will these also work for engineered tissues (most likely 3D)?
These may be able to be done on 3D but may also have to be varied and are very
hard so its very improbable.
38) What are some of the additional characteristics of engineered tissues that we
might like to analyze?
We can analyze viability, toxicity, compatibility to host tissues, etc.
39) What is your working definition of a biomaterial? Give some examples of
biomaterials.

A biomaterial is a material implanted in body or material that contacts body fluids


outside of body. Examples of biomaterials include medical scaffolds and contact
lenses.
40) Based on what you know about native tissues, what might some desirable
features of biomaterials for tissue engineering be?
Some desirable features of biomaterials are biodegradability, biocompatibility with
the native tissue, and bioresorbability.
41) What are some of the key aspects of a cells environment (that we might want
to manipulate for our own goals and/or to keep the cells alive and well)? (There
should be A LOT of these)
We may want to manipulate the pH, temperature, salinity, concentration, O2
concentration, mechanical environment, etc.
42) What are some cell behaviors that we might want to attempt to
manipulate/affect?
We might want to manipulate cell motility, cell signaling, cell proliferation, etc.
43) What are different ways that cells communicate with each other and their
environment?
Cell-cell recognition, chemical signals, hormones, etc.
44) What components should we add to our engineered tissues to make them
perfect?
We should add reproducibility, complexity, structural integrity, etc to make them
perfect.
45) What are some of the limitations to successfully engineering tissues today?
Some of the limitations include the difficulty of reproducibility of tissue samples, the
difficulty of preparation and attribution of a molecular response to the original donor
tissue are some limitations to successful tissue engineering today.
46) What are some of the design principles guiding TE?
Reproducibility, viability, cost, utility, functionality, marketability, etc are all design
principles.