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MCTE > Exam 1: Tissue Morphology/Cells & Culturing > Flashcards

Exam 1: Tissue Morphology/Cells & Culturing Flashcards

1
Q

What is embryogenesis?

A

-Starts just after fertilization
-Cells go through cleavage (division, but different in that the cells don’t increase in volume, just in number)
-Upon reaching uterus, gastrulation occurs and organs start to develop.

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2
Q

What are blastomeres?

A

New cells arise from early divisions. They are todipotent-capable of forming an entire organism.

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3
Q

What is a blastocyst?

A

-Forms after multiple divisions, pre-implantation
-has a blastocele (fluid filled cavity), trophoblast(surrounds blastocele, forms the placenta) and an internal cell mass (pluripotent cells that will form the entire embryo)
-the internal cell mass from a blastocyst can be isolated; it’s the source of embryonic stem cells.

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4
Q

Blastula

A

Cells migrate to establish 3 germ layers:
Gastrolation is occurring, endoderm moved in. Ectoderm encloses the others. Mesoderm elongated. A new pocket forms (archenteron will be the gut
-Neurulation is also occurring during gastrolation, forms mural tube that will become the spinal cord and brain. Neural crest is created. Technically a fourth germ layer.

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5
Q

What are the three germ layers?

A

Ectoderm-outer skin, parts of eye, pituitary gland
Mesoderm-connective tissue, heart &blood vessels, bone, muscle, kidney, cartilage
Endoderm-liver, pancreas, thyroid, bladder lining, respiratory tract
Neural Crest Cells-neurons, glial cells

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6
Q

Nucleus

A

Control center that houses DNA, controls cell division, growth, protein production, & cell death

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7
Q

Ribosomes

A

produce proteins by assembling ammino acid sequences according to the genetic code

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8
Q

Mitochondria

A

Mitochondria are the site of respiration and the ‘powerhouses’ of cells, pumping out energy which is then stored in ATP (adenosine triphosphate).

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9
Q

Endoplasmic Reticulum

A

The rough ER is studded with ribosomes, giving it a bumpy or ‘rough’ appearance. It folds and tags newly-synthesized proteins before transporting them to wherever they are needed in the body. The smooth ER does not have ribosomes attached to it and is instead involved in hormone and lipid synthesis.

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10
Q

Golgi Apparatus

A

a series of flatted, membrane-bound sacs that packages and distributes substances to the outer cell membrane, where they either become part of the lipid bilayer or leave the cell.

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11
Q

Lysosomes

A

small, spherical organelles that are packed full of digestive enzymes. Their key function is to break down and recycle unwanted material for the cell, such as old cell parts or invading bacteria and viruses. Lysosomes also play an important role in apoptosis

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12
Q

Cytoplasm

A

jelly-like substance that fills the interior space of cells. It is mainly composed of water, but also contains salts, enzymes, and other organic molecules. The cytoplasm surrounds and protects the organelles of the cell and is where many cellular processes (such as protein synthesis and glycolysis) take place

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13
Q

Plasma Membrane

A

semipermeable phospholipid bilayer. The cell membrane controls which substances enter and leave the cell, and also separates the interior of the cell from its external environment

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14
Q

Cytoskeleton

A

a structure that helps cells maintain their shape and internal organization, and it also provides mechanical support that enables cells to carry out essential functions like division and movement. There is no single cytoskeletal component. Rather, several different components work together to form the cytoskeleton
Microtubules are the largest type of filament, with a diameter of about 25 nanometers (nm), and they are composed of a protein called tubulin. Actin filaments are the smallest type, with a diameter of only about 6 nm, and they are made of a protein called actin. Intermediate filaments, as their name suggests, are mid-sized, with a diameter of about 10 nm. Unlike actin filaments and microtubules, intermediate filaments are constructed from a number of different subunit proteins

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15
Q

What happens when cells differentiate?

A

they attain a
specialized, mature characteristic form
* Different shape & size
* Different molecular content of cytoplasm

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16
Q

Basic Tissue Types:

A

Epithelium - continuous 2D sheet of cells, dont move individually, (blood vessel, skin, bladder, kidney lining)
Connective tissue - offer mechanical support, arise from the mesoderm, (bone, ligaments)
Muscle - smooth, cardiac, skeletal
Nervous tissue - CNS- spinal cord & brain, PNS

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17
Q

Autonomous Specification

A

-internally endowed with capacity to achieve fate, doesn’t need outside stimulus.

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18
Q

Conditional Specification

A

-depends on interactions with other cells or materials

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19
Q

Induction: Methods in which cells influence each other

A

Diffusion of soluble signals, contact w/ ECM, direct cell-cell receptor contact.

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20
Q

Induction: Types

A

Negative- collective of cells that restrict potential of each other
Instructive- A responder cell changes due to interaction
Permissive - Responder cells have all potential but require environment
Reciprocal - tissues signal each other
(NIPR)

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21
Q

Cell Adhesion

A

Adhesion molecules enable specific binding
between surface of a cell and neighboring cells
or extracellular matrix
* Integrins – primarily involved in adhesion to
extracellular matrix
* Cadherins – involved in cell-cell adhesion

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22
Q

Cell Migration

A

Requirements
* Generation of mechanical force
* Traction with another surface
Very related to adhesion

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23
Q

Invagination

A

Cell tissue moves inwards to form a bean shape from a sphere

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24
Q

Involution

A

Tissue compresses on either side of a sphere to form a bubble

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25
Q

Epiboly

A

tissue moves in from the side to fill the interior of a sphere

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26
Q

Delamination

A

tissue separates horozontally

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27
Q

Diffusion of Soluble Signals

A

Diffusion of signaling molecules, or morphogens, creates spatial
gradients
§ Cells sense their position in the gradient and develop accordingly

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28
Q

Constraints on Morphogenesis

A

Number of cells that participate
§ Physical dimensions of spontaneous cellular
rearrangements
§ Time scale over which morphogenesis takes
place

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29
Q

Types of Cells

A

Undifferentiated cells
* Responsible for replenishing old, injured, or dead
cells
§ Differentiated cells
* Specialized cells that perform a unique function in
the body

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30
Q

Properties of Stem Cells

A

Self renewal and differentation

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31
Q

Stem Cell Self Renewal:

A

Symmetric Division- daughter cells have identical genetic makeup, can differentiate
Asymmetric division- one daughter copy, one new different cell-can be caused by intrinsic mechanisms: localization of cell polarity an desegregation of cell fate determinants, or extrinsic mechanisms: signals from ECM such as spacal and soluble factors

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32
Q

todipotent

A

Generates every cell in the body including the
placenta and extra-embryonic tissue
Can form the entire human being,

from zygotes, cannot self renew

33
Q

pluripotent

A

Can generate every cell in the body except the
placenta and extra-embryonic tissue
Cannot form the entire human being

embryonic stem cells from blastocyst inner cell mass, self renewing

34
Q

Multipotent

A

broad potential, self renewing

35
Q

differentiated

A

non mitotic, functional

36
Q

Types of Differentiation

A

Dedifferentiation- undoing dif.
Redifferentiation- undone, then redone
Transdifferentiation- moving from one specialization to another

37
Q

Epigenetics

A

Stable alterations in gene expression potential that
arise during development without altering the DNA
sequence
* Regulated by:
§ DNA methylation - cytosines get methyl groups, methylation patterns passed on to daughter cells during division
§ Histone modifications - histones provide structural support for chromosomes, modification makes chromatin more condensed, less genes available for transcription, also caused by methyl groups

38
Q

Types of Stem Cells:

A

Embryonic, Adult, and induced pluripotent

39
Q

Embryonic Stem Cells

A

Embryonic stem cells are derived from inner cell mass blastocyst
* Can self-renew indefinitely in culture
* Embryonic stem cells used in research are made
in a petri dish, not in a woman’s body
Needs a feeder layer
Advantages
* Indefinite proliferation capacity
* Pluripotency
§ Shortcomings
* Mouse origin
* Human origin
§ Culture conditions
§ Immune rejection
§ Purification of homogenous cell population
§ Ethical concerns

40
Q

Adult stem cells

A

undifferentiated cells,
which reside in tissue/organs
* Hematopoietic stem cells: blood cells, Found in:
* Bone marrow
* Umbilical cord blood
* Peripheral blood
§ Produces all red and white
blood cells, platelets
§ HSC are identified by the
surface marker CD34
* Mesenchymal stem cells: bone and muscle

41
Q

iPSC(induced pluripotent)

A

isolate tissue, reprogram and wait, the cells become stem cells
Advantages:
* Cells would be genetically identical to patient or
donor of skin cells (no immune rejection!)
* Do not need to use an embryo
§ Shortcomings:
* Cells would still have genetic defects
* One of the pluripotency genes is a cancer gene
* Viruses might insert genes into places where we
don’t want them (causing mutations)

42
Q

ES Cell markers

A

OCT-4 (transription factor)
Alkaline phosphatase
Telemerase

43
Q

Where can we harvest cells?

A

Autograft, Allograft, Xenograft

44
Q

Harvesting Methods:

A

§ Bone Marrow Aspirate
* Only need local anesthesia (in upper hip or sternum)
* Easily accessible
* Use large needle + heparinized syringe (to stop blood clot)
typically yielding about 2 mL of marrow
* Marrow contains mesenchymal stem cells, hematopoietic
cells, adipocytes, endothelial progenitor cells
§ Uroepithelial Cell Flush-out
* No anesthesia required
* Method involves repeated filling/emptying with isotonic
salt solution
§ Tissue Biopsies
* Skin; take a biopsy punch
* Cartilage; arthroscopy to harvest tissue
* Heart/Liver/Kidneys (etc.) can use fluoroscopy (to guide to
location) and catheter (to retrieve sample)

45
Q

Tissue Disassociation techniques

A

Cells from tissue biopsies must be dissociated from each other and
the ECM for growing in tissue culture plates
Mechanical Disruption
* Vortex with a digestion buffer
* Pipet vigorously
* Dice with a scalpel
* Enzymatic Disruption
* Disrupt the Cell-ECM connections
* Trypsin, proteases, collagenases, papain,
dispase
* Disrupt Cell-Cell connections
* Chelating agents (such as EDTA) to
disrupt cadherins
* Explanted Tissues
* May or may not require digestion or
disruption. Cells may migrate outward on their
own.

46
Q

Cell sorting: Selective adhesion

A

Isolate desired cell types by
manipulating adhesion characteristics
§ Isolated based on differences based in:
* Cell surface receptors
* E.g., Coat TC plate with protein that some cells will bind to stronger than
others
* Adhesion speed
* E.g., Remove cell suspension at a given time interval
§ Pros: Does not require staining or special equipment
§ Cons: Limited to highly active cells
Cell Selection: Antibody Driven

47
Q

Cell Sorting: Antibody Driven

A

Select a target antigen on cell surface
>Magnetic-Activated Cell-Sorting: Label cells with
magnetic bead-conjugated antibodies and separate with
strong magnetic field
>Fluorescence-Activated Cell
Sorting (FACS):
* Label cells with fluorescently
conjugated antibody
* Flow through machine to sort
fluorescent vs. non-fluorescent

48
Q

HeLa Cells

A

first cells successfully cultured
in vitro
* Cervical cancer cells
* Highly proliferative/aggressive/hard to kill off

49
Q

Cell Culture Media Components

A
  1. Water
    * Pure water is needed
  2. Balanced Salts (NaCl, CaCl2, KCl, NaHCO3)
    * Provide a physiologic ionic environment
    * Maintains intra- and extra-cellular osmotic balance
    * Physiological osmolality: ~285 mOsm/kg H2O
  3. Energy Molecules (Glucose)
    * Glucose most common substance
    * Average amount of glucose added is ~5g/L
    * Other energy substrates are also often
    included (sodium pyruvate)
  4. Buffering Agents
    * Role is to maintain the pH of
    cells (typically at 7.2-7.4)
    * Carbonates, phosphates, and
    citrates are examples of
    buffering agents
    * Most common buffer system is
    bicarbonate buffer system
  5. pH Change Indicators
    * Phenol Red (yellow acidic, purple basic)
  6. Anti-fungal/bacterial substances
    * Prevents Infections
    * Ex: Penicillin/Streptomycin; Gentamicin
    * Not always added since they can influence cells
    (e.g., altered cell metabolism and increased stress)
  7. Amino Acids (AA)
    * Essential AAs cannot be synthesized by the cells
    themselves, so must be added to media
  8. Vitamins
    * Most common vitamins in media include Bvitamins (thiamin, folic acid, etc.) and
    Vitamin C
  9. Growth Factors
    (depends on which type of cells culturing)
    * Fetal Bovine Serum
    o ~10% added to cell culture media
    o Provides a wide variety of
    macromolecular proteins, nutrients,
    hormones
    * Custom Growth Factor Cocktails
    o For endothelial cells, would include
    VEGF, bFGF, etc.
50
Q
  • Conditions the incubator maintains
    for mammalian cell growth:
A

37°C
* 95% Humidity
* 5% CO2

51
Q

Flask Names

A

T-Flask (T-#), where # = growth area in cm2
* Petri Dish (P-#), where # = growth area in cm2

52
Q

cell proliferation is constrained by what factors?

A
  • Cell density
  • Nutrient availability
  • Waste production
53
Q

Cell Signaling

A

: initiated by the generation of a
ligand, which is produced by a sending cell to
alter the physiology of a receiving cell.

54
Q

Types of Signaling:

A

Differentiation
Proliferation
Protein Secretion
*and migrate and die

55
Q

Signaling depends on…

A

Ligand concentration & other ligand type exposures/environmental factors

56
Q

Types of ligands

A

§ ECM molecules
§ Membrane-bound ligands
§ Diffusible molecules

57
Q

Soluble Signals

A

§ Cytokines: Small molecules (5-20 kDa) that
influence cell-cell interactions & cell behavior
§ Chemokines: A subset of small molecular
weight cytokines that stimulate migration
§ Mitogen: Small molecules that promote mitosis
§ Growth factors: substances capable of
stimulating cell proliferation, maturation, and/or
differentiation

58
Q

Receptor

A

Receptor: transmembrane protein
* Interacts with ligand/signal in extracellular space
* Transduces signal into cell

59
Q

Autocrine

A

the production and secretion of an extracellular mediator by a cell followed by the binding of that mediator to receptors on the same cell to initiate signal transduction.

60
Q

Paracrine

A

a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells

61
Q

Endocrine

A

the signaling molecules (hormones) are secreted by specialized endocrine cells and carried through the circulation to act on target cells at distant body sites

62
Q

Signal Transduction

A

During transduction, the signal is amplified
One receptor-ligand interaction leads to the
activation of many downstream proteins
Binding>enzymatic activity>protein modification>second messenger>proteolysis>protein relocation

63
Q

Adhesion impacts:

A

Migration
* Cell-cell aggregation
* Cell & molecular transport

64
Q

Cadherins

A

Cadherins: transmembrane
proteins that mediate cell-cell
adhesion
* Homophilic binding
* Requires Ca2+
§ Examples:
* E-cadherin
* P-cadherin
* S-cadherin

65
Q

Ig-Like Receptors

A

Cell surface receptors mediate
cell-cell adhesion
* Homophilic binding
* Does not require Ca2+
§ Example:
* NCAM (Neural Cell Adhesion
Molecule)

66
Q

Selectins

A

Transmembrane proteins that
mediate cell-cell adhesion
* Heterophilic binding

67
Q

Integrins

A

Heterodimer - α and β sub-units
§ Extracellular domain binds ECM
protein
§ Cytoplasmic domain connects
cytoskeletal proteins
§ Bind to small peptide fragments

68
Q

ECM Composition

A

Proteoglycans: protein-sugar complex- Complex that consist of a
protein molecule to which
chains of GAGs are covalently
attached. -Glycosaminoglycans (GAGs):
* High molecular weight
polysaccharides, which are
usually highly sulfated and thus
negatively charged

§ Proteins: extended, fibrous
§ Other: growth factors, digestive enzymes, etc.

69
Q

Collagen

A

Constitutes >25% of all protein
in humans
* High tensile strength
* More than 20 different types
* Basic structure: alpha helical
chains formed by interaction of
three polypeptides
* Fibrillar collagens – collagen
monomers are staggered and
crosslinked.

70
Q

Fibronectin

A

Dimer: two similar
polypeptides that are
covalently linked together
* Subunits linked by disulfide
bonds
§ Each chain contains
binding sites for:
* Other ECM components
* Cell surface receptors

71
Q

Structural Proteins

A

Elastin – gives elasticity, found in tissues that
undergo repeated stretching (e.g., blood vessels)

72
Q

Adhesive Proteins

A
  • Laminin – adhesive peptides RGD, YIGSR, IKVAV
  • Tenascin – adhesive and anti-adhesive properties
  • Entactin- found in basement membranes in
    association with laminin, contains RGD
  • Thrombospondin – RGD sequence
  • Vitronectin – present in blood vessels & other tissues,
    RGD sequence
73
Q

Focal Adhesions

A

Integrin-containing structures that
form mechanical links between intracellular actin
bundles and the extracellular substrate

74
Q

How does the ECM influence adhesion mediated signaling?

A

ECM composition (which proteins are present) dictate
integrin binding
§ ECM concentration
§ ECM mechanical properties (stiffness/elasticity)
§ Protein confirmation/variants
Extent of cell spreading (i.e., cell area)
§ Formation of actin filament bundles
§ Presence of focal adhesions

75
Q

Non-reproducible measurement of adhesion

A

Detaching cells by washing
§ Detaching cells through “flicking”

76
Q

Cell migration

A
  1. Lamellipodia protrude
    * Increased number of actin
    polymerization
  2. Adhesion to the matrix
    through integrins
    * Provides a link to actin
    cytoskeleton
  3. Contraction of cytoplasm
    by myosin-based motors
    * Expressed as a traction force
    on substrate
  4. Rear release and
    displacement
    * Enzymatic processes break
    the complex or the
    membrane rips leaving
    integrins behind
  5. Recycling of remaining
    integrins
    * Integrins transported to
    leading edge
77
Q

Optimal adhesion

A

Biphasic dependence
§ Weak adhesion
* New attachments can’t form
at leading edge
§ Strong adhesion
* Rear attachments can’t release

78
Q

Modes of Migration

A

Chemotaxis: Movement along a soluble chemical concentration gradient
§ Haptotaxis: An insoluble chemical concentration gradient
§ Durotaxis: Substrate stiffness gradient
Galvanotaxis: Electric current
§ Contact Guidance: Surface topography
§ Contact Inhibition:
Lamellipodium inhibited by neighboring adjacency

MCTE (2 decks)

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