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In vitro Methods for the Evaluation of

Biomaterials

Dr. Anil Kumar PR

Scientist F and Head of Division of Tissue Culture
Department of Applied Biology, Biomedical Technology Wing

20-03-2021

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Content
• Definitions
• Basic cell culture
• Standards
• Cytotoxicity testing
– Biological Evaluation of Medical Devices —Part 5: Tests for
in vitro Cytotoxicity
• Cytocompatibility testing
• Research profile Division of Tissue culture

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Definitions
• Biomaterial
– A biomaterial is "any substance (other than drugs) or
combination of substances synthetic or natural in origin,
which can be used for any period of time, as a whole or as
a part of a system which treats, augments, or replaces any
tissue, organ, or function of the body".
• Biocompatibility
– The ability of a material to perform with an appropriate
host response in a specific application
– Host Response — The response of the host organism (local
and systemic) to the implanted material or device.

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Biocompatibility
The quality of not having toxic or injurious effects on biological
system
Dorland's Medical Dictionary

The ability of a material to perform with an appropriate host

response in a specific application
The Williams Dictionary of Biomaterials

Comparison of the tissue response produced through the close

association of implanted candidate material to its implant site
within the host animal to that tissue response recognized and
established as suitable with control materials.
American Society for Testing and Materials.
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Biocompatibility
Refers to the ability of a biomaterial to perform its desired
function with respect to a medical therapy, without eliciting any
undesirable local or systemic effects in the recipient or the
beneficiary of that therapy, but generating the most appropriate
beneficial cellular or tissue response in that specific situation,
and optimizing the clinically relevant performance of that
therapy.
On the Mechanisms of Biocompatibility and Revisiting the Definition of
Biocompatibility

Division of Tissue Culture 5

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Using biomaterials
• The materials to be used in vivo have to be approved by
the apex regulatory authority.
• The material has to go through a series of
“biocompatibility” tests.
– Cytotoxicity
– Hemolysis
– Acute systemic toxicity
– Sub chronic/chronic
– Oral toxicity
– SensitizationIntravenous toxicity
– Mutagenicity, Genotoxicity
– Pyrogenicity

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Cell culture
• Denotes the growing of cells in vitro, including the culture of
cells in experimental system which artificially reproduces the
experimental conditions necessary to guarantee the viability
of cells or tissues from a living organism

• Implies the tissue or outgrowth from a primary explant is

dispersed (mechanically or enzymatically) into a cell
suspension, which may then be cultured as an adherent
monolayer or a suspension.

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(Quality System as per ISO 17025)

Floor Plan

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Basic equipment
• Biosafety cabinet or
Laminar flow bench
• Incubator
– Temperature 37±2 C
– CO2 5%
– Relative humidity 95%

• Microscope
– Inverted phase contrast
microscope

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Basic equipment
• Deep Freezer (-85C)
• Autoclave
• Membrane filtration units
• Microplate reader
• Refrigerator
– Culture media
– Growth factors
– Enzymes
• Tissue cultureware
– Culture bottles
– Multiwell plates

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Aseptic requirement
Mycoplasma
• Class 100 (working area)
• Contamination free culture
Routine air monitoring
Bacterial, fungal
Staining or PCR +ve (Sigma)

Mycoplasma

-ve (L-929)

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Types of cell culture

• Primary culture
– Organ culture
– Explant culture
– Disaggregated cells
• Culture of established cell line
• Histotypic culture
• Organotypic culture

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Primary cell culture

• Cells when surgically or enzymatically removed from an
organism and placed in suitable culture environment will
attach and grow are called as primary culture
– Explant culture
• An outgrowth of cells arising from a simple small fragment (primary
explant) of tissue that adheres to the growth surface.
– Organ culture
• The primary explant culture where the histological structure and the
associated differentiated properties or original tissue is maintained.
• Air lift culture.

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Primary cell culture

• Disaggregated cells
– When a tissue sample is
disaggregated, either mechanically
or enzymatically.
– Cell suspension transferred to
culture dish will enter into different
phases.
– Proliferation of cells in culture
requires attachment to a substrate
and occurs until limited by cell-to-
cell contact, resulting in formation
of cellular monolayer (confluency).
– Passage or Subculture : Harvest of
cells from monolayer to new
surface.

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Primary cell culture

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Culture of cell line

• Those cells arises from primary culture that are capable of
proliferation at the time of first successful subculture is
called a cell line.
• The term cell line implies that cultures from it consist of
lineages of cells originally present in the primary culture.
• The cell line could be finite or continuous
• Cells can be genetically modified (immortalized) to obtain
continuous or established cell line.
– A culture which is apparently capable of an unlimited number of
population doublings; often referred to as an immortal cell culture.

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Growth curve

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Selection of tests- Duration & Nature of Contact

ISO 10993-1

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Different standards for in vitro cell culture methods for
Biomaterials - Cytotoxicity & Cytocompatibility
1. ISO 10993-5, 2009. Biological evaluation of Medical Devices-
Part5. Tests for in vitro cytotoxicity

2. ISO 7405:2008. Dentistry - Evaluation of biocompatibility of

medical devices used in dentistry - Test methods for dental
materials- Part 6.2 and 6.3
Annex B Dentine barrier cytotoxicity test

3. ASTM F 1027-86 (2002) Standard practice for assessment

of Tissue and Cell compatibility of orofacial Prosthetic
materials and devices

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Different standards for in vitro cell culture methods for
Biomaterials - Cytotoxicity & Cytocompatibility
4. ASTM F813-01 (2002) Standard Practice for Direct Contact
Cell Culture Evaluation of Materials for Medical Devices
5. ASTM F895-84 (2001) Standard Test Method for Agar
Diffusion Cell Culture Screening for Cytotoxicity
6. NF S 91-142 - Dental implants. Cytocompatibility. Study of
Cellular proliferation
7. NF S 91-143- Dental implants. Cytocompatibility. Study of
total cellular proteins

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Different standards for in vitro cell culture methods for
Biomaterials - Cytotoxicity & Cytocompatibility
8. NF S 91-144- Dental implants. Cytocompatibility.
Evaluation of 51Cr extracellular release
9. NF S 91- 145 Dentistry - Dental Implants Cytocompatibility
Study of cells attachment and spreading on the biomaterial
10. NF S 91-146 Dental implants- Cytocompatibility
Study of cellular multiplication, migration and adherence
11. USP 28 (2005) -87 -Biological Reactivity tests, In Vitro
12. Biomaterials & Biomolecules for TEMPS- ASTM 2005

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In vitro cytotoxicity testing

• ISO 10993- Part 5
– Describes test methods to assess the in vitro cytotoxicity of
medical devices.
– These methods specify the incubation of cultured cells in
contact with a device and/or extracts of a device either
directly or through diffusion.
diffusion
– These methods are designed to determine the biological
response of mammalian cells in vitro using appropriate
biological parameters.

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Cytotoxicity
• An effect or range of effects, microscopic and/ or visual, linked
to the presence of test materials. These range from
impairment in growth to complete cell lysis or dissolution
when test cultures are compared to the reference blank.
• Test sample
– Material, device, device portion, component, extract or portion
thereof that is subjected to biological or chemical testing or evaluation
• Controls
– Positive : Material provides a reproducible severe cytotoxic response
• The ZDEC and ZDBC polyurethanes, Phenol
– Negative : Material does not produce a cytotoxic response
• UHMWPE, HDPE

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Sterility of test samples

• Sterilization (ISO 11135-1)
– “a validated process used to render a product free from viable
microorganisms”
• Sterility of test samples
– Samples should be sterile and handled aseptically throughout the test
procedure
– The effect of sterilization methods or agents on the device should be
considered in defining the preparation of the test sample prior to use in
the test system
• Methods of sterilization
– Steam sterilization (autoclaving): 15-20 min, 15 psi, 121 C
– Radiation : 60Co gamma rays, ultra violet C (germicidal)
– Ethylene Oxide (EtO gas) : 180 min, low temperature (37 C, 50-60 C)
– Hydrogen peroxide (low temperature): 45 – 70 min,H2O2 Plasma, 45-50 C
– Membrane filtration: Liquid samples, 0.22 um pore filter.

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Preparation of material for cytotoxicity tests

• Liquid test samples
– Tested by either by direct deposition or deposition on a biologically
inert absorbent matrix (Filter discs).
• Absorbent test samples
– Test samples that are absorbent shall be soaked with culture medium
prior to testing to prevent adsorption of the culture medium in the
testing vessel.

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Cells
• Established cell lines are preferred and where used
shall be obtained from recognised repositories.
• American Type Culture Collection (ATCC) are
endorsed by ISO
– CCL 1 (NCTC clone 929),
– CCL 163 (Balb/3T3 clone A31),
– CCL 171 (MRC-5) and CCL 75 (WI-38),
– CCL 81 (Vero) and
– CCL 10 [BHK-21 (C-13)]
– V-79 379A

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Continuous cell lines used for general or basal cytocompatibility

Mouse L-929 Subcutaneous connective tissue fibroblast

P1534 Leukemia cells
3T3 Connective tissue fibroblasts

Monkey Vero Kidney fibroblasts

Human HeLa Cervical carcinoma cells

MRC-5 Embryonic lung fibroblasts
Hep-G2 Hepatocarcinoma
Caco-2 Adenocarcinoma cells
MCF-7 Breast carcinoma

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Methods Of Cytotoxicity Testing - (ISO 10993-5)

• Test by Direct Contact
– Qualitative
• Test on Extract - Short term or long term
– Quantitative
• Test by Indirect Contact
– Filter Diffusion / Agar Diffusion

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Methods Of Cytotoxicity Testing - (ISO 10993-5)

Annexure
• Neutral Red Uptake Cytotoxicity
• Colony Formation Cytotoxicity Test
• MTT Cytotoxicity Test
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• XTT Cytotoxicity Test
Sodium 3´-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-
nitro) benzene sulfonic acid hydrate

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Test by Direct Contact

• Changes in general morphology, vacuolisation,
detachment, cell lysis, cell density and membrane
integrity

• Form of test samples

– Materials that have various shapes, sizes or physical states (i.e.
liquid, gels, solids, etc.) may be tested without modification in
the cytotoxicity assays.
– The preferred test sample of a solid material should have at
least one flat surface. If not, adjustments shall be made to
achieve flat surfaces.

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Test by Direct Contact

• Changes in general morphology, vacuolisation,
detachment, cell lysis, cell density and membrane
integrity
None cytotoxic Severe cytotoxic

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Cytotoxicity by Direct Contact Method

Reactivity grades for agar and filter diffusion test and direct contact test

0 hour 24 hour Neutral red staining to show cells

in direct contact are viable

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Indirect Contact Test

• Similar to Direct contact – Also known as Agar diffusion
• Mix fresh culture medium containing serum with melted agar - 0.5 % to 2
%
– Agar should be suitable for the growth of mammalian cells in culture.
– The agar/culture medium mixture should be in a liquid state and at a
temperature that is compatible with mammalian cells.
• Place replicate test sample on the solidified agar layer
• Incubate for 24 h to 72 h and examine the cells to determine cytotoxic
effect
• Use of a vital stain can aid in the detection of cytotoxicity , e.g. Neutral
Red.

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Test on Extract
• The cytotoxic effect of extracts of the test devices to
the cell layer.
• Evaluation
– Qualitative
• The malformation, degeneration and lysis of cells are observed
using microscope.
– Quantitative
• Measure cell activity (MTT assay)

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Test on Extract

Biomaterial

Extraction
Vehicle

Qualitative (Microscopy)

Extraction
Conditions
Test on Extract

Quantitative (Bio Assay)

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Extraction vehicles
• Extracting conditions should attempt to simulate or
exaggerate the clinical use conditions.
• Due to the nature of certain materials (e.g.
biodegradable materials), alteration of the chemical
structure can occur during the extraction procedure.
• Extraction vehicle
– The extraction vehicle(s) has to be selected considering the
chemical characteristics of the test sample.
– Culture medium with serum;
– Physiological saline solution;
– Other suitable vehicles
– Purified water and Dimethyl Sulfoxide (DMSO

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Extraction conditions
• Extraction shall be conducted under one of the following conditions
and shall be applied according to the device characteristics and
specific conditions for use
• Extraction conditions
a. (24 ± 2) h at (37 ± 1) °C;
b. (72 ± 2) h at (50 ± 2) °C;
c. (24 ± 2) h at (70 ± 2) °C;
d. (1 ± 0,2) h at (121 ± 2) °C.
• For medical devices that are in short-term contact (no greater than
4 h cumulative contact duration) with intact skin or mucosa and
that are not implanted, this may include extraction times of less
than 24 h but no less than 4 h, as given in a) to c).

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Cytotoxicity by Test on Extract - Grading

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Cytotoxicity by Test on Extract - Observations

None cytotoxic Slight cytotoxic Mild cytotoxic

Metal (Stent) Polymer (PVC) Polymer (Composite)

Moderate cytotoxic Severe cytotoxic

Polymer (PET modified) Polymer (Composite)

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Cytotoxicity by Test on Extract – Image Analysis

100% 50% 25%

2. Mild 2. Mild 1. Slight

Neg Pos Round Spread Total Round %

25% 68 185 253 26.88

50% 117 202 319 36.68

100% 165 210 375 44.00

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Quantitative Evaluation of Test on Extract

• MTT assay (Measuring cell activity)
Calorimetric assay to measure activity of mitrocondrial
enzyme to reduce MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

Solubilize
(Acid alcohol)

Formazan Read
(Insoluble)
MTT
(Soluble)

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Quantitative Evaluation of Test on Extract

Cytotoxicity of different phenol concentration by MTT assay

120
100 100
100
96
% cell activity

80
73
60
40
28
20
0
1.3 g/L 0.65 g/L 0.325 g/L 0.162 g/L Control
% Concentration of Phenol

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Cell activity by MTT Assay

Drugs and powder materials

120

100
10
% cell activity

80 5
2.5
60
1.25
40 0.625
0.3125
20
0.15625
0
CPC SS
Con (mg/ml)

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GENERAL APPLICATION OF CYTOTOXICITY TESTS

• Screening of new materials
 New product components (polymers, fabrics, adhesives
etc)
 Batch testing of
 Raw Materials
 Finished Products

Candidate packaging materials to see the transfer of

toxic moieties from package to product (eg: paper,
plastics, coatings and ink)

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General application of cytotoxicity tests

• Material validation of new product designs
• Trouble shooting to determine cause of both
developmental and production problems
• ETO dissipation
• Comparison of in vitro and in vivo as a means of
prediction
• To measure TC ID 50, to get an idea of the minimum safe
dose
• In vitro accelerated system
• Quality control

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Cytotocompatibility Tests
• Cytotoxicity vs Cytocompatibility
– Cytotoxicity involves negative criteria such as cellular
alterations, cell death, hampered growth etc.
– Cytocompatibility evaluates positive criteria
• A material will be considered as cytocompatible if
both structure and functions of the tissue in direct
contact with it remain unchanged
– Depends directly upon the quality of the material surface
– Individual parameters will be analyzed separately

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General Cytocompatibility
• Relates to function common to all cells like
– Cell death
– Absence or change in metabolic activity
– Lysis of cells
– Cell adhesion
– Alterations in cellular morphology
– Reduced cellular proliferation

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Neutral Red Uptake

PVC [Stabilized] High Density Poly Ethylene Test sample

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Cell Viability

Confocal image showing viability of L-929 cells adhered on Composite Material

FDA + PI staining

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Membrane integrity

Fluorescein Di Acetate and Propidium Iodide staining of L-929 cells

Acridine orange and ethidium bromide staining of L-929 cells on materials

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Cell adhesion

Osteoblasts adhesion on Ti HepG2 adhesion on hydrogel

HaCaT adhered on PCL-Chitosan

Nanofibers

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Cell morphology

L-929 SIRC

Hepatocytes Sinusoidal Endothelial cells

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Cytoskeletal organization

Actin staining of L929 cells Actin staining of L929

Even distribution
on TCPS of actin cytoskeleton indicatescells
goodoncell spreading
copolymer
CK 3/12 Actin

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Cell attachment rate and doubling time

Tritiated thymidine uptake assay (n=3)

Cell attachment = count of total number of cells – count of unattached cells
log(2)
Doubling time  T0 – initial time point
K N  T1 – final time point
log 1  N0 – cell number at T0
Where, K  2.3   N0  N1 – cell number at T1
T1 - T0

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Cell cycle analysis

• Eg
– Cell cycle of L929 was not
affected after growing on
the copolymer
– Percentage of dead cells
(Gate ‘A’) on TEST was
comparable to Control

Cell cycle analysis of L929 cells after culturing for 24

hours a) Control, b) TEST

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Functional cytocompatibility using specialised cells

• Depending on functionality of material with respect
to end use.
Cell type Function

Lymphocytes/Macrophages Immunomodulatory Activity

Endothelial cells Modulation of coagulation

Osteoblasts Bone forming activity

Corneal cells Cytoadhesive capability

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Testing nanomaterials
• ASTM E2526 – 08
Standard Test Method for Evaluation of Cytotoxicity of
Nanoparticulate Materials in Porcine Kidney Cells and Human
Hepatocarcinoma Cells.
– Nanoparticulate test materials in suspension in cell culture
media and appropriate controls are added to cell cultures.
– The release of LDH indicates membrane damage and the
diminution of MTT reduction indicates loss of cell viability.
– These are quantitative indicators of cytotoxicity. Aseptic
procedures are required.

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Processing Cells, Tissues, and Organs for Use in Tissue
Engineered Medical Products
• F2210 – 02 (Reapproved 2010)
This guide describes the processing, characterization,
production, and quality assurance of cells, tissues,
and organs used for Tissue Engineered Medical
Products (TEMPs).
• It concerns aspects of processing activities for cells,
tissues, and organs to be further processed.

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In Vitro Rat Hepatocyte DNA Repair Assay

• ASTM - E1397 – 91 (Reapproved 2008)
In Vitro Rat Hepatocyte DNA Repair Assay
• DNA repair is an enzymatic process that involves the recognition
and excision of DNA-chemical adduct followed by DNA strand
polymerization and ligation to restore the original primary structure
of the DNA.
• This process can be quantitated by measuring the amount of
labeled thymidine incorporated into the nuclear DNA of cells that
are not in S-phase and is often called unscheduled DNA synthesis
(UDS).
• The primary rat hepatocyte DNA repair assay has proven to be
particularly valuable in assessing the genotoxic activity and
potential carcinogenicity

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Advantages and disadvantages

• Advantages • Disadvantages
– Extremely sensitive – Simplified version
– Physiological environment – Oversensitive
controllable – Short life span
– Cellular level
– Low concentration
– No wound reaction
– Many variables and replicates
– Legal, moral and ethical questions
– Human cells
– Large amount of homogenous cells
– Tissue Engineered Products
– Stem Cells

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Conclusions
• Pertinent data on biocompatibility
• Results together with histology and other analyses gives
exclusive picture of biological performance
• Good correlation between in vitro & in vivo
• More sensitive
• Rarely harmful in vivo due to corrosion products,
secretory enzyme & biological millieu
• Recent Trends
• Tissue Engineered constructs to simulate in vivo situation

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