ISO 19090:2018
(Main)Tissue-engineered medical products — Bioactive ceramics — Method to measure cell migration in porous materials
Tissue-engineered medical products — Bioactive ceramics — Method to measure cell migration in porous materials
ISO 19090:2018 specifies the test method to be followed for measuring and documenting the cell migration ability of porous bioactive ceramic materials. ISO 19090:2018 is not applicable to porous materials that have low or no cell adhesion properties, for instance synthetic polymers and metals. These types of materials will require longer times to allow effective transfer and migration of cells from the cultured substrate to the test specimen. To minimize influences of cell passages, cell kinds, differences in cell culture consumables including culture medium and fetal bovine serum etc., the method uses a porous bioactive ceramics, which is clinically and widely used in each country, as a reference material for calculation of relative migration distance.
Produits médicaux issus de l'ingénierie tissulaire — Céramiques bioactives — Méthode de mesure de la migration cellulaire dans les matériaux poreux
General Information
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 19090
First edition
2018-01
Tissue-engineered medical
products — Bioactive ceramics —
Method to measure cell migration in
porous materials
Produits médicaux issus de l'ingénierie tissulaire — Céramiques
bioactives — Méthode de mesure de la migration cellulaire dans les
matériaux poreux
Reference number
ISO 19090:2018(E)
©
ISO 2018
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ISO 19090:2018(E)
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ISO 19090:2018(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test specimen . 3
6 Procedures . 3
7 Calculation .11
7.1 Mean and standard deviation .11
7.2 Relative mean and standard deviation by normalization .12
8 Test report .13
Annex A (informative) Results of tests for determination of test conditions .14
Annex B (informative) Results of international round robin test .26
Bibliography .33
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ISO 19090:2018(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
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expressions related to conformity assessment, as well as information about ISO's adherence to the
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URL: www.iso.org/iso/foreword.html.
ISO 19090 was prepared by Technical Committee ISO/TC 150, Implants for surgery, Subcommittee SC 7,
Tissue-engineered medical products.
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ISO 19090:2018(E)
Introduction
“Bioactive ceramics” are widely used in orthopaedic and dental fields due to their bioactivities and
bioaffinities. Porous bioactive ceramics are designed as bone void fillers, and cell migration from tissue
into their pores is an expectation for effective repair of bone defects; thus, they are one of the promising
candidates for cell scaffolds for bone tissue engineering medical products.
To clarify the clinical safety and usefulness of these bioactive ceramics, physical, chemical and biological
properties must be examined. In the methods used, animal tests are the ultimate and essential methods
to examine biological properties of bioactive ceramics; however, numbers of both animals and animal
[3]
tests must be reduced under the concept of 3R (Replacement, Reduction and Refinement) .
The first and most important property for porous biomaterials including bioactive ceramics is cell
migration capability, because cell proliferation, differentiation, tissue formation and tissue maturation
in and surroundings of porous biomaterials do not occur without cell migration.
Currently, two different cell-seeding methods are used for estimating “cell migration” property: One is
dropping a cell suspension on the top surface of a porous material. This method tests the penetration
ability of the “cell suspension” under gravity and estimates the number of cells that migrate into and are
held within the porous material. The other method is shaking a porous material in the cell suspension.
This method also tests the penetration ability of the “cell suspension” like the above method but uses
shaking to drive the cells into the porous scaffolds. Both methods test the abilities of cell penetration
and retention only, and do not test the intrinsic ability of the cell to migrate simulating what happens
in vivo. Body fluid itself can sufficiently carry cells across a minor gap between the implanted material
and the host bone. Accordingly, no cell migration test methods have been reported that mimic cell
behaviour in vivo.
When porous bioceramics are implanted into bone defect, cells migrate into the pore to form new bone.
In this process, migration of osteoblasts mainly plays important roles for osteoconduction. That is to
say, no osteoconduction nor bone formation can occur without osteoblast migration.
Therefore, it is imperative to establish a quantifiable method to measure cell migration potential of
porous bioactive ceramics in a manner similar to how cells behave in vivo, in order to evaluate their
potential appropriately as materials for tissue-engineered medical products.
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INTERNATIONAL STANDARD ISO 19090:2018(E)
Tissue-engineered medical products — Bioactive ceramics
— Method to measure cell migration in porous materials
1 Scope
The document specifies the test method to be followed for measuring and documenting the cell
migration ability of porous bioactive ceramic materials.
This document is not applicable to porous materials that have low or no cell adhesion properties, for
instance synthetic polymers and metals. These types of materials will require longer times to allow
effective transfer and migration of cells from the cultured substrate to the test specimen. To minimize
influences of cell passages, cell kinds, differences in cell culture consumables including culture medium
and fetal bovine serum etc., the method uses a porous bioactive ceramics, which is clinically and widely
used in each country, as a reference material for calculation of relative migration distance.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 10993-5, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-5 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
bioceramics
ceramics used that enhance biological functions when implanted in the human body
3.2
bioactive ceramics
bioceramics (3.1) with direct bone bonding property when implanted into bone defect
3.3
biomaterial
material used in or to be used in medical and dental field
3.4
full confluent
cell cultured dish is almost completely (≈95 % to 100 %) covered with a monolayer of cells
3.5
complete medium
cell culture media that is recommended for the chosen cell type by the supplier of the cells with all
required supplements cell culture medium that is confirmed by the user that the cells used in the test
proliferate well without any mutation
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ISO 19090:2018(E)
3.6
osteoblast-like cell
established cell lines which widely recognised to have “osteogenic activity”
3.7
cell group
group of cells composed of at least five cells where the distance between each cell is less than the
cell width
4 Principle
The cell migration property of porous biomaterials has been estimated by measuring cell numbers that
are seeded in the porous body by two methods, however, they measure penetration of cell suspension
and cell attachment, not migration by cells themselves.
The present method is very simple and effective to measure cell migration by themselves. Cells
confined in a confluent layer of a culture dish are able to further migrate and proliferate from this layer
into the porous bioceramic materials that are placed on top of the culture dish as shown in Figure 1.
To minimize influences of cell passages, cell types, differences in cell culture consumables including
culture medium and serum, etc., the method uses a reference material that porous bioactive ceramics
commercialised and used clinically with good clinical results in each country for calculation of relative
migration distance.
a) Before: No cells exist in a test material b) After: Cells move up to a test material
through pores
Key
1 culture dish filled with cell culture medium
2 cells adhered on a bottom of culture dish
3 test material
Figure 1 — Schematic drawing of test method
After the initial transfer of cells onto the material interface, they start to migrate into pores of the
materials. This migration distance will differ in relation to the materials properties and chemical
stability, surface morphologies, and pore structures similar to what is seen in vivo and which mimics
the initial part of the bone regeneration process.
Giemsa staining is a very stable and easy method to stain cells for this method.
Linear longest migration distance of a valid cell measured from the cross section of the Giemsa stained
porous bioactive ceramics is well reflected cell migration in vivo.
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ISO 19090:2018(E)
5 Test specimen
5.1 Shape and dimensions
The shape of the specimen should be a disk (10 ± 0,2) mm in diameter and (2 ± 0,1) mm in thickness.
5.2 Number of test specimens
At least five pieces are required for the comparison between two (reference and test) materials.
5.3 Reference material
Commercially available porous bioactive ceramics in each country that are confirmed as good bone void
filler by basic researches and/or clinical results. The reference material available in multiple countries
is recommended.
6 Procedures
6.1 Measurement of thickness and diameter of specimen
The thickness and diameter of specimen shall be measured with a calliper or a micrometer.
6.2 Sterilization of test specimens
Specimens shall be sterilized by a method which does not affect the material quality before the test.
6.3 Deaeration of test specimens
Specimens shall be immersed in 5 mL complete medium in a centrifuge tube which can be sealed tightly
with a lid. After an 18 to 21 gauge needle connected with a 20 mL syringe is inserted through the lid of
the tube, the specimens shall be deaerated by pulling the plunger of the syringe back completely with
tapping on the tube for 2 min to 3 min to eliminate bubbles.
6.4 Cell culture
Osteoblast-like cells shall be seeded on 6-well cell culture plates or ⌀35 mm cell culture dishes and
cultured to full confluency. Seeding cell number will be decided by the preliminary test to obtain
confluency within 3 days. MG-63 and MC3T3-E1 are recommended for osteoblast-like cells. To obtain
4 4
confluent cells within 3 days in 6-well culture plate, seeding of 6,0 × 10 per well for MG63 or 8,0 × 10
per well for MC3T3-E1 will be needed. Cells shall be observed under a phase-contrast microscope for 2
days to 3 days after seeding to check cell confluency and to avoid cell overconfluency.
NOTE The number of cells is counted by conventional methods using the hemocytometer, described in
ISO 13366-1 or cell counting devices, described in ASTM F2149–01.
The number of cells to be seeded to reach full confluency within 3 days should be confirmed by the
preliminary test, if the cell line other than recommended in this document will be used in this test.
When wells (dishes) are covered with confluent cells, 3 wells (dishes) shall be stained with Giemsa
to record the cell confluent status before migration test as follows: After being fixed with 2 %
glutaraldehyde overnight, Giemsa solution with appropriated concentration shall be prepared with
PBS, and 2,5 mL of the solution shall be added to each well (dish) and incubated for 3 min at room
temperature. Then the staining solution shall be removed and washed with 2,5 mL distilled water
for 3 times. Stereoscopic micrograph of each well or dish shall be taken at x10 or maximum available
magnification so that the bottom of plates or dishes can be observed in the same scope.
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ISO 19090:2018(E)
6.5 Placing specimens on the cell layer
When cells have reached full confluency, one deaerated specimen shall be placed on the cell layer and
the SUS316 stainless steel double ring with 10 mm in ring outer diameter and 0,8 mm to 1,0 mm in wire
diameter, shown in Figure 2, shall be put on the specimen to brace in place. Both shall be placed with
forceps without dropping nor agitation of the medium. There can be some roughness and unevenness
on the surface of the specimen due to manufacturing process. Choose a smooth surface of the specimen
to place on the cell layer.
When transferring the plates (or dishes) into the CO incubator, take care not to move the specimen
2
inadvertently since it could impair the close contact of the specimen with the cell layer. The cells shall
be cultured another 3 days without medium change or any other procedure.
Figure 2 — Stainless steel double ring
Set the weight on the sample to ensure close contact between the sample and the confluent cell layer.
However, if the influence of the extra weight on the cell is unclear, the acute cytotoxicity should be
evaluated by cell proliferation assay as follows, to ensure that there is no effect.
a) wash the weight with ultrapure water with applying ultrasonic for 1 h followed by rinsing with
ultrapure water 3 times.
b) dry heat sterilize the weight at 160 °C for 3 h.
c) place the weight on the full confluent cell layer as same as the cell layer to be used in the test and
observed cell shape and numbers surroundings of the weight with phase-contrast microscope daily
for at least 3 days.
6.6 Treatments after cell culture
The specimen shall be harvested 3 days after incubation. To prevent detachment of cells from the
specimen, place the bottom side (cell contact surface) of the specimen up after harvesting. The
specimen shall be washed with PBS for 3 times and fixed with 2 % glutaraldehyde overnight. After
fixation, the specimen shall be washed with PBS for 3 times and immersed in 10 mL of Giemsa solution
with appropriate concentration at room temperature. After 3 min, the sample shall be transferred to
another vessel and washed with 10 mL of distilled water. The wash is repeated 3 times.
If this staining method does not work or is very different from the manufacturer’s instruction, follow
the staining protocol in the manufacturer’s instruction.
Wells or dishes after harvesting specimen shall be washed with PBS for 3 times and fixed with 2 %
glutaraldehyde overnight. After fixation, the wells or dishes shall be washed with PBS for 3 times and
pour 2,5 mL of Giemsa solution with appropriate concentration at room temperature. After 3 min,
the solution will be removed and washed with distilled water. The wash is repeated 3 times. Then,
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ISO 19090:2018(E)
the staining solution shall be removed and washed with 2,5 mL of distilled water for 3 times. Cell
migrations from the edge of the cell transferred areas on the well or dish are observed, if the specimen
and stainless steel double ring are not affected cell viability. Further, if cells in the specimen placed area
showed no change in shape and numbers but did not transfer well, low “flatness” of the specimen can be
the reason.
6.7 Positive and negative controls
Including positive and negative controls for the assay signal can be useful for each assay. For example,
an appropriate negative control can be to conduct the measurement in the absence of cells. The scaffolds
can be placed in dishes without cells, incubated with culture medium, washed, stained, imaged and
scored. As an example of an appropriate positive control, the scaffolds can be directly seeded with cells
using a pipette, incubated in medium to let the cells adhere (possibly 4 h or 24 h), washed, stained,
imaged and scored.
These controls provide assurance that the assay is working effectively and help with interpreting the
results. In the case where cells migrate into the test scaffolds, it is important to demonstrate that the
negative controls did not score positively for cell migration. This provides evidence that the background
staining of the scaffold and the scoring procedure, among other things, are reliable. In the case where
there is poor migration into the test scaffolds, it is important to demonstrate that the positive controls
scored positively for cells. This provides evidence that cells were viable, the stain was effective, and the
scoring procedure was reliable (among other things). Including positive and negative controls in the
assay makes it possible to interpret the results and improves confidence in the conclusions.
6.8 Observations of cells that migrated into a specimen
Annexes A and B show typical results and are useful to understand practical results and procedures as
referred in the following procedures.
The cell-contact (bottom) side of the specimen shall be observed with a stereoscopic microscope.
Stereoscopic microphotographs of the bottom side shall be taken. The specimen shall be cut into two
pieces using a thin scalpel commonly used for eye surgery. Depending on the flatness of bottom surface
of the specimen, stained area might have irregular distribution and shape as shown in Figures A.5
and B.3; therefore, the cutting position shall be determined from the stained specimen interface
(bottom) that had been in direct contact with the cell layer; and the incision shall be made along the
longest determined length through the darkest stained area of the specimen (Figures 3 to 6).
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ISO 19090:2018(E)
Key
A cutting position
B stained area
C non-stained area
Figure 3 — Schematic drawing to determine cutting line for specimen surface of which stained
area is more than a half
Key
A cutting position
B stained area
C non-stained area
Figure 4 — Schematic drawing to determine cutting line for specimen surface of which stained
area is less than a half
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ISO 19090:2018(E)
Key
A cutting position
B stained area
C non-stained area
Figure 5 — Schematic drawing to determine cutting line for specimen surface of which stained
areas are partitioned
Key
A cutting position
B stained area
C non-stained area
Figure 6 — Schematic drawing to determine cutting line for specimen surface of which stained
areas are distributed
The cross section should be observed preferably with a stereoscopic microscope. Stereoscopic
microphotographs of the cross section of the specimen and micrometre at the same magnification shall
be taken.
NOTE 1 Specimens are cleaved with a scalpel that is normally used for eye surgery. If it is impossible to cleave
specimens with a scalpel, the specimen is cut with a diamond cutter or similar apparatus using phosphate
buffered saline as lubricant.
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ISO 19090:2018(E)
NOTE 2 The same specimen stained with the same Giemsa staining solution after soaking in the same cell
culture medium without cells for the same period can be used as a control to confirm background staining level
of the specimen to clarify the stained parts of the test specimens are cell(s) or not.
NOTE 3 Fluorescent labelling of cells and observation with a confocal microscope can be used for the following
longest migration distance measurement as well, if confocal microscope is available in the laboratory.
6.9 Measurement of migration distance of cells in a specimen
6.9.1 General
All measurements should be performed preferably under a stereoscopic microscope at x10 and x20
(objective x1 and x2, eye-piece x10) magnification.
6.9.2 Maximum migration distance of cell
The longest linear distance from the bottom side of the specimen to the furthest stained cell or cell
group shall be measured for each specimen. Before measurement, the following shall be checked.
a) Using a stereoscopic microscope at high magnification (at least x40), the cellular nature of the
staining shall be validated using general cell criteria including nuclear staining and morphological
features showing cellular process and attachment to the surface of the porous structures.
b) Validation of cell and cell group for measurement described below is based on the results from
multiple inter-laboratory tests. The following empirical validation avoids inclusion of a cell or cell
group that can have lodged non-specifically within the larger pore sites as a result of cell culture
media changes and washings prior to fixation.
The followings are the protocol to validate cell and cell group in detail.
Longest linear length from bottom side to top end of a cell or cell group, which observed most far from
bottom side, shall be measured.
6.9.2.1 Determination of linear longest migration distance for cell group
As shown in Figure 7, the top of a cell group is continued from the bottom side; thus, the white line is
measured as the linear longest distance for a cell group.
Key
grey area stained area with many cells
NOTE The white line is the longest linear distance.
Figure 7 — Measurement point of migrated distance in a simple stained area
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ISO 19090:2018(E)
Key
grey area stained area with many cells
A single cell
NOTE The white line is the longest linear distance.
Figure 8 — Measurement point of migrated distance in a simple stained area with a cell but not
a cell group
Key
grey area stained area with many cells
NOTE The white line is the longest linear distance.
Figure 9 — Measurement point of migrated distance in partitioned stained area
In case 1, the cell is divided from the top of the cell group as shown in Figure 8, the dark line indicates
linear longest distance to a “cell”, but not to a cell group; thus, the linear longest distance to a “cell
group” is the white line.
Even stained area is divided as shown in Figure 9, the higher stained part is measured as a linear
longest distance for a cell group as indicated as the white line. Thus, if no cell group divided from
bottom stained area are observed as in Figure 7, the linear longest distance of the “cell group” is defined
as white lines in Figure 7.
When cell groups are divided from bottom stained area, the following validation shall be performed.
In case 1 (Figure 10), the closest distance from the cell group A to the cell group that already is
confirmed as a valid cell group, length of line b, is shorter than that of line a, migration distance of
valid cell group; thus, A is confirmed as a valid cell group. In this case, no divided cell group with longer
distance from bottom side. Accordingly, the linear longest distance to cell group is a length of line c.
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ISO 19090:2018(E)
Key
A cell group
a migration distance of a valid cell group
b distance between cell group A and a valid cell group
c linear longest distance of cell group
Figure 10 — The measurement point of migration distance in stained area when a cell group
migrate irregularly in specimen at very near to the stained area
In case 2 (Figure 11), start from the cell group C, which is the closest cell group from bottom side.
Length of line i, distance to the closest valid cell group from the C, is shorter than that of line j; thus the
C is a valid cell group and the length of line h is valid migration distance as the same as the cell group
A and line c in the case one. However, much longer distances from the other cell group D to bottom,
length of line m, are also valid due to the same reason, i.e., length of line k is shorter than that of line
l. Contrarily, cell group B is not valid, because the length of line g, the closest distance to the valid cell
group, is longer than that of line h.
6.9.2.2 Determination of linear longest migration distance for cell
After determined linear longest migration distance for the “cell group,” the same validation will be
performed for a cell, using the closest “cell group” not “cell.” For instance, in Figure 11, when all circles,
B, C and D, are “cells”, not “cell groups”, the cell B shall be validated using lines e and f instead of lines g
and h. Therefore, even if length of line g is shorter than that of line h, cell B is not valid because length of
line e is longer than that of line f. When C is a “cell group”, using lines g and h for validation of the cell B.
After validations mentioned above, the longer distance between linear longest distances of the cell and
cell group shall be chosen as the maximum cell migration distance for the specimen.
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ISO 19090:2018(E)
Key
B, C, D irregularly migrated cell gro
...
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