Cell and Tissue Survival Assays
This presentation is the property of its rightful owner.
Sponsored Links
1 / 50

Cell and Tissue Survival Assays PowerPoint PPT Presentation


  • 140 Views
  • Uploaded on
  • Presentation posted in: General

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.

Download Presentation

Cell and Tissue Survival Assays

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Cell and tissue survival assays

Cell and Tissue Survival Assays

Lecture 6


Cell and tissue survival assays

  • 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


Cell and tissue survival assays

  • 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


Cell and tissue survival assays

Fate of irradiated cells

Division Delay-(Dose = 0.1 to 10 Gy)

Interphase Death-Apoptosis

Reproductive Failure-Loss of clonogenicity


Cell and tissue survival assays

Measurement of Killing

A.In-vitro

1.Plating Efficiency

2.Survival

B.In-vivo

1.Xenografts

2.In-situ tumors

3.Survival


Cell and tissue survival assays

In-vitro clonogenic assays

  • Clonogenic assays – techniques in which the endpoint

  • observed depends directly on the reproductive integrity

  • of individual cells.

  • These systems are directly analogous to cell survival

  • in vitro.


Cell and tissue survival assays

In-vitro clonogenic assay

Tissue (tumor) sample

Chop it up + trypsin

Single cell suspension

Culture dish with medium at 370 C

1 – 2 Weeks

Colonies


Cell and tissue survival assays

In-vitro clonogenic assay


Cell and tissue survival assays

  • 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


Cell and tissue survival assays

Reproductive Death

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


Cell and tissue survival assays

MULTIPLICITY

  • 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.


Cell and tissue survival assays

Survival Curves for Mammalian cells

First in-vitro survival curve was reported in 1956


Cell and tissue survival assays

Surviving fraction for cells irradiated

to 6 Gy

C3H cells C3H cellsV-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 0.28

1 Gy x 6 fractions 0.36 0.33 0.34

2 Gy x 3 fractions 0.52 0.55 0.65

3 Gy x 2 fractions 0.11 0.20 0.14

6 Gy x 1 fractions 0.06 0.10 0.08

Smith et al, IJROBP1999


Cell and tissue survival assays

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.


Cell and tissue survival assays

Radiation sensitivity profiles for cells of human origin


Cell and tissue survival assays

  • 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


Cell and tissue survival assays

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.


Cell and tissue survival assays

Clones re-growing in-situ

Skin colonies


Cell and tissue survival assays

Clones re-growing in-situ

Skin colonies


Cell and tissue survival assays

Clones re-growing in-situ

Skin colonies


Cell and tissue survival assays

Clones re-growing in-situ

Crypt cells of the

mouse jejunum


Cell and tissue survival assays

Clones re-growing in-situ

Crypt cells of the

mouse jejunum


Cell and tissue survival assays

Clones re-growing in-situ

Crypt cells of the mouse jejunum


Cell and tissue survival assays

Clones re-growing in-situ

Crypt cells of the

mouse jejunum


Cell and tissue survival assays

Clones re-growing in-situ

Testes stem cells


Cell and tissue survival assays

Clones re-growing in-situ

Testes stem cells


Cell and tissue survival assays

Clones re-growing in-situ

Testes stem cells


Cell and tissue survival assays

Clones re-growing in-situ

Kidney tubules


Cell and tissue survival assays

Clones re-growing in-situ

Kidney tubules


Cell and tissue survival assays

Cells transplanted to another site

Bone-marrow

stem cells


Cell and tissue survival assays

Cells transplanted to another site

Bone-marrow stem cells

Photograph of a spleen showing the colonies to be counted


Cell and tissue survival assays

Cells transplanted to another site

Bone-marrow stem cells


Cell and tissue survival assays

Cells transplanted to another site

Mammary cells


Cell and tissue survival assays

Cells transplanted to another site

Thyroid cells


Cell and tissue survival assays

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

normal tissues

Note the substantial

range of radiosensitivities

with shoulder width being

the principal variable


Cell and tissue survival assays

  • 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


Cell and tissue survival assays

Functional endpoints

Dose-response relationships

Pig skin


Cell and tissue survival assays

Functional endpoints

Dose-response relationships

Pig skin


Cell and tissue survival assays

Functional endpoints

Dose-response relationships

Pig skin


Cell and tissue survival assays

Functional endpoints

Dose-response relationships

Rodent skin


Cell and tissue survival assays

Functional endpoints

Dose-response relationships

Rodent skin


Cell and tissue survival assays

Functional endpoints

Early and late response of the lung based on breathing rate.

Breathing frequency increases progressively

with dose after a treshold of about 11 Gy


Cell and tissue survival assays

Functional endpoints

Spinal 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.


Cell and tissue survival assays

Functional endpoints

Spinal cord myelopathy

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


Cell and tissue survival assays

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 the

length of the cord irradiated in the rat.


Cell and tissue survival assays

Functional endpoints

Dose-response relationships

The 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.


Cell and tissue survival assays

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


  • Login