cell and tissue survival assays

Ahmed GroupAhmed GroupLecture 6Lecture 6

Cell and Tissue Survival Assays

Lecture 6


• In vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints


• In-vitro clonogenic assays• Calculation of plating efficiency and surviving fraction• In-vivo clonogenic assays• Bone marrow stem cell assays, jejunal crypt stem cell assay, skin clones, kidney tubules• Functional endpoints


1. Division Delay - (Dose = 0.1 to 10 Gy)2. Interphase Death - Apoptosis3. Reproductive Failure - Loss of clonogenicity

Fate of irradiated cells


Measurement of Killing

A. In-vitro

1. Plating Efficiency2. Survival

B. In-vivo

1. Xenografts2. In-situ tumors3. Survival


In-vitro clonogenic assays

• Clonogenic assays – techniques in which the endpointobserved depends directly on the reproductive integrityof individual cells.

• These systems are directly analogous to cell survivalin vitro.


Tissue (tumor) sample

Chop it up + trypsin

Single cell suspension

Culture dish with medium at 370 C

1 – 2 Weeks

Colonies

In-vitro clonogenic assay


In-vitro clonogenic assay


Reproductive DeathLoss of proliferation ability of cell exposed to radiation, as assessed by colony forming assay.


• The influence of cellular multiplicity (number of cells per potential colony-forming unit) on the determination of radiation sensitivity can be evaluated for a range of multiplicity distributions.

• Cell surviving fraction can be calculated using no multiplicity correction, an average multiplicity correction or the fractional distribution of multiplicities of the control and irradiated population.

• Multiplicity corrections are required when the number of cells per potential colony-forming unit is greater than 1.00 either immediately after plating or at the time of irradiation.

• Both the control and irradiated populations must be corrected for multiplicity. • Multiplicity errors are most pronounced in the low-dose range, e.g. in the survival range

with 2 Gy. • The error introduced by using an average vs fractional distribution of multiplicities

increases with the multiplicity dispersion. • Seemingly small errors due to uncorrected multiplicity effects lead to markedly different

predicted isoeffect doses when amplified through multiple (e.g. 30) fractions.

MULTIPLICITY


Survival Curves for Mammalian cells

First in-vitro survival curve was reported in 1956


Surviving fraction for cells irradiated to 6 Gy

C3H cells C3H cells V-79 cells(plateau phase) (Exponential) (plateau phase)Treatment

Controls (0.37) (0.34) (0.59)0.3 Gy x 20 fractions 0.30 0.24 0.281 Gy x 6 fractions 0.36 0.33 0.342 Gy x 3 fractions 0.52 0.55 0.653 Gy x 2 fractions 0.11 0.20 0.146 Gy x 1 fractions 0.06 0.10 0.08

Smith et al, IJROBP1999


Parameters of survival curves

PE: Plating efficiency. Percentage of cells able to form colonies

Dq: The quasi-threshold dose for a given population that often measures the width of the shoulder

D0: The dose that reduces the surviving fraction to e-1 (= 0.37) on the exponential portion of the curve or the dose that produces 37% survival.

n: Extrapolation number. This value is obtained by extrapolating the exponential portion of the curve to the abscissa.


Radiation sensitivity profiles for cells of human origin


In-vivo clonogenic assays

- The techniques developed by Withers and his colleagues are based on the observation of a clone of cells regenerating in situ in irradiated tissue. In situ re-growth techniques include the skin, crypt cells in the jejunum or colon, testes stem cells, and kidney tubules

- Systems in which cell survival is assessed by transplantation into another site include bone-marrow stem cells, thyroid cells, and mammary cells

The various types of normal tissue assay systems are described in following slides.


Clones re-growing in-situ

Skin colonies



Crypt cells of the mouse jejunum


Testes stem cells



Testes stem cellsClones re-growing in-situ


Testes stem cells



Kidney tubules



Cells transplanted to another site

Bone-marrow stem cells




Photograph of a spleen showing the colonies to be counted



Mammary cells



Thyroid cells


Summary of dose-response curves for all of the clonogenic assays in normal tissues

Note the substantial range of radiosensitivitieswith shoulder width beingthe principal variable


Functional endpointsDose-response relationships

Pig skin


Pig skin



Rodent skin



Functional endpointsDose-response relationshipsRodent skin


Functional endpointsEarly and late response of the lung based on breathing rate.

Breathing frequency increases progressivelywith dose after a treshold of about 11 Gy


Functional endpointsSpinal cord myelopathy

A dose-response relationship can be determined for late damage caused by local irradiation of the spinal cords of rats. After latent periods of 4 to 12 months, symptoms of myelopathy develop: palpable muscle atrophy, followed by impaired use of hind legs

Fractionation and protraction. Dose per fraction is very important, with the dose to produce paralysis increasing dramatically with number of fractions.


Functional endpointsSpinal cord myelopathy

Fractionation and protraction. The effect of a large number of very small fractions is shown.


Functional endpoints Spinal cord myelopathy

Volume effects. The total volume of irradiated tissue has an influence on the development of tissue injury. Shown is the relation between tolerance dose and thelength of the cord irradiated in the rat.


Functional endpointsDose-response relationshipsThe parameters of the dose-response curve for any normal tissue system for which a

functional endpoint can be observed may be inferred by performing multifractionexperiment and estimating the α/β ratio. Because α/β is the dose at which cell killing bylinear and by quadratic components are equal, the implication is that dose-response relationships for late-responding tissues are “curvier” than for early-responding tissues.This is important in the discussion of fractionation in radiotherapy.


Summary- In vitro clonogenic assays – techniques in which the endpoint observed

depends directly on the reproductive integrity of individual cells.

- In vivo clonogenic endpoints include systems in which cells re-grow in-situ and some in which cells are transplanted to another site.

- Dose-response curves can be obtained as a result of clonogenic assays.

- The radiosensitivity of cells from normal tissues varies widely. The width of the shoulder of the curve is the principal variable.

- Dose-response curves for functional endpoints, distinct from cell survival, can be obtained for:

- The shape of the dose-response relationship for functional endpoints, obtained from multifraction experiments, is more pertinent to radiotherapy than clonogenic assays

- The ratio α/β may be inferred from multifraction experiments in systems scoring nonclonogenic endpoints

•pig skin and rodent skin by measuring skin reactions•early and late response of the lung by measuring breathing rate•spinal cord by observing myelopathy

cell and tissue survival assays

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