ASTM F2315-03

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:F2315–03
Standard Guide for
Immobilization or Encapsulation of Living Cells or Tissue in
Alginate Gels
This standard is issued under the fixed designation F2315; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Encapsulation in insoluble alginate gel is recognized as a rapid, non-toxic, and versatile method for
immobilization of macromolecules and cells. Microencapsulated cells or tissue as artificial organs are
under study for treatment of a variety of diseases such as Parkinson’s disease, chronic pain, liver
failure, hypocalcemia, and, perhaps the most well-known example, immobilization of islets of
Langerhans utilized as an artificial pancreas in the treatment of diabetes. Since alginates are a
heterogeneous group of polymers with a wide range of functional properties, the success of an
immobilization or encapsulation procedure will rely on an appropriate choice of materials and
methodology. This must be based on knowledge of the chemical composition of alginate and the
correlation between the structure, composition, and functional properties of the polymer, as well as
differences in gelation technologies. It is also important to recognize the need for working with highly
purified and well-characterized alginates in order to obtain gels with reproducible properties.The aim
of this guide is to provide information relevant to the immobilization or encapsulation of living cells
and tissue in alginate gels.
1. Scope F748 Practice for Selecting Generic Biological Test Meth-
ods for Materials and Devices
1.1 This guide discusses information relevant to the immo-
F1251 Terminology Relating to Polymeric Biomaterials in
bilization or encapsulation of living cells or tissue in alginate
Medical and Surgical Devices
gels. Immobilized or encapsulated cells are suitable for use in
F1903 Practice for Testing For Biological Responses to
biomedical and pharmaceutical applications, or both, includ-
Particles in vitro
ing, but not limited to, Tissue Engineered Medical Products
F1904 Practice for Testing the Biological Responses to
(TEMPs).
Particles in vivo
1.2 This guide addresses key parameters relevant for suc-
F1905 Practice For Selecting Tests for Determining the
cessful immobilization and encapsulation in alginate gels.
Propensity of Materials to Cause Immunotoxicity
1.3 This standard does not purport to address all of the
F1906 Practice for Evaluation of Immune Responses In
safety concerns, if any, associated with its use. It is the
Biocompatibility Testing Using ELISATests, Lymphocyte
responsibility of the user of this standard to establish appro-
Proliferation, and Cell Migration
priate safety and health practices and determine the applica-
F2064 Guide for Characterization and Testing of Alginates
bility of regulatory requirements prior to use.
as Starting Materials Intended for Use in Biomedical and
2. Referenced Documents
Tissue-Engineered Medical Products Application
2.2 USP Document:
2.1 ASTM Standards:
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved Sept. 10, 2003. Published October 2003. DOI:
10.1520/F2315-03.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2315–03
USP Monograph USP 24/NF19 Sodium Alginate delivery devices. These products may be appropriate for
2.3 Other Referenced Documents: implantation based on supporting biocompatibility and physi-
EN-ISO 10993 Biological Evaluation of Medical Devices cal test data. Recommendations in this guide should not be
International Conference on Harmonization (ICH) S2B interpreted as a guarantee of clinical success in any tissue
Genotoxicity: A Standard Battery for Genotoxicity Test- engineered medical product or drug delivery application.
ing of Pharmaceuticals (July 1997)
5. Gelation Techniques
3. Terminology
5.1 Most methods for encapsulation of cells or tissue in
3.1 Definitions: alginate gels basically involve two main steps. The first step is
3.1.1 alginate, n—a polysaccharide obtained from some of the formation of an internal phase where the alginate solution
the more common species of marine algae, consisting of an containingbiologicalmaterialsisdispersedintosmalldroplets.
insoluble mix of calcium, magnesium, sodium, and potassium In the second step, droplets are solidified by gelling or forming
salts. Alginate exists in brown algae as the most abundant a membrane at the droplet surface.
polysaccharide, mainly occurring in the cell walls and inter- 5.2 The most simple and common way to produce small
cellularspacesofbrownseaweedandkelp.Itsmainfunctionis beads or capsules is by forming droplets of a solution of
to contribute to the strength and flexibility of the seaweed sodium alginate containing the desired biological material
plant. Alginate is classified as a hydrocolloid. The most (cells, tissues, or other macromolecules) and then exposing
commonly used alginate is sodium alginate. them to a gelling bath. A gelling bath may be a solution
2+ 2+
3.1.2 APA bead, n—alginate-poly-L-lysine-alginate bead.
containing divalent cross-linking cations such as Ca ,Sr ,or
2+ 2+
3.1.3 encapsulation, n—a procedure by which biological Ba . Monovalent cations and Mg ions do not induce
materials,suchascells,tissues,orproteins,areenclosedwithin
gelation (34).
a microscopic or macroscopic semipermeable barrier. 5.3 Concentration of Ions:
3.1.4 endotoxin, n—pyrogenic lipopolysaccharides derived
5.3.1 The concentration of gelling ions used must be deter-
from bacterial cell walls, usually associated with membrane mined based upon factors such as desired gel strength, type of
protein unless purified. Though endotoxins are pyrogens, not
alginate used (G- or M-rich), and isotonicity of the gelling
all pyrogens are endotoxins. solutions. Calcium ion concentrations of from 50 to 150 mm
3.1.5 gel, n—the three-dimensional network structure aris-
are often used.
2+ 2+
ingfromintermolecularpolymerchaininteractions.Suchchain 5.3.2 Other gelling ions may be used, such as Ba or Sr .
2+
interactions may be covalent, ionic, hydrogen bond, or hydro-
The concentration of Ba in the gelling solution must be
phobic in nature. See also Terminology F1251. determined based upon the desired characteristics of the final
2+
3.1.6 immobilization, n—the entrapment of materials, such gel and on regulatory and toxicological considerations as Ba
as cells, tissues, or proteins within, or bound to, a matrix. can induce toxic effects in cells.
3.1.7 pyrogen, n—any substance that produces fever. 5.3.3 Concentration of Non-gelling Ions—Various additives
3.2 Additional definitions regarding alginate may be found present in the gelling solution that do not participate in the
in Guide F2064. Additional definitions regarding polymeric formationofcross-linksconstitutenon-gellingions.Theseions
+
biomaterials may be found in Terminology F1251. may be Na , which can be used to produce homogeneous gels
(see 7.1), ions present in cell culture medium (if present in the
4. Significance and Use
gelling bath), and others.
4.1 The main use is to immobilize, support, or suspend
6. Formation of Beads
living cells or tissue in a matrix. The use of an encapsulation/
immobilization system may protect cells or tissues from 6.1 Bead size is one of the most important parameters of
immune rejection. When immobilizing biological material in
alginategelbeadsandcapsulesinbiomedicalapplications.The
alginate gels, there are numerous parameters that must be
appropriate size will often be a compromise. The bead itself
controlled. This guide contains a list of these parameters and
must be large enough to contain the biological material. Larger
describes the methods and types of testing necessary to
beads are also easier to handle during washing or other
properly characterize, assess, and ensure consistency in the treatments. In many applications involving cells, the cells
performance of an encapsulation system using alginate. This
should be homogeneously distributed within the internal cap-
guide only covers single gelled beads, coated or not, and not sularmatrix.Whengeneratingbeads,thedesiredmeansizeand
double capsules or other constructs.
acceptable size distribution should be accounted for. The size
4.2 The alginate gelation technology covered by this guide of the beads is primarily controlled by regulating droplet
may allow the formulation of cells and tissues into biomedical
formation.
devices for use as tissue engineered medical products or drug 6.2 Droplet Size—Droplet size is dependent upon several
factors: The size of the material to be immobilized or encap-
sulated (that is, single cells or cell aggregates such as pancre-
Available from U.S. Pharmacopeia (USP), 12601Twinbrook Pkwy., Rockville,
atic islets), the technique used to generate droplets (that is,
MD 20852.
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
4th Floor, New York, NY 10036, http://www.ansi.org.
5 6
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758, The boldface numbers in parentheses refer to the list of references at the end of
1211 Geneva 13, Switzerland. this standard.
F2315–03
pipette or syringe, coaxial air flow, electrostatic generator, apparent viscosity of an alginate solution may change depend-
jet-cutter, and so forth) and the viscosity of the alginate ing upon whether the alginate is dissolved in water or in a
solution. Generally, for biomedical applications, droplet size is salt-containing medium. When using droplet generators, size
regulated to give a gelled bead having a diameter of <200 to and sphericity of the beads will, therefore, depend on the
1000 µm. Per unit volume, smaller beads yield a larger surface viscosity of the alginate solution and the distance the droplets
area to transplant volume, a ratio that results in enhanced fall before reaching the gelling solution. In addition, the final
survival of tissue due to better nutritional and oxygen supply. size of the beads will be dependent of the gelling conditions
Varioustechniquescanbeusedtoformdropletsasdescribedin used.
more detail by Dulieu et al. (11). These include, but are not 6.4 Concentration of Biological Material (Cells or
limited to: Others)—In applications involving immobilization of cells
6.2.1 Extrusion through a Needle—Beads can be made by diffusionpropertiesofdifferentmoleculeswithinthebeadswill
dripping an alginate solution from a syringe with appropriate also depend strongly on the load of cells.As a consequence of
diameter needle directly into a gelling bath. While this method diffusion limitations cells surrounded by other cells within the
does not require any instrumentation, the size and size distri- gel network may, therefore, be strongly influenced by the
bution of the produced beads are difficult to control. metabolism of the surrounding cells. As a result surrounded
6.2.2 Coaxial Air or Liquid Flow—The coaxial air jet cells may be trapped in a micro-environment lacking essential
system is a simple way of generating small beads (down to nutrients like oxygen. This may typically result in cell death in
around400µm),althoughthesizedistributionwillnormallybe the center of the beads with an outer rim of viable cells.
larger as compared to an electrostatic system. In this system, a 6.5 Presence of Impurities—Several authors (12, 38) found
coaxialairstreamisusedtopulldropletsfromaneedletipinto that perfectly spherical and smooth alginate beads could only
a gelling bath (Fig. 1). be formed by using a highly purified alginate.
6.2.3 Electrostatic Potential—An electrostatic potential can
7. Final Capsule or Bead Properties
be used to pull droplets from a needle tip into a gelling bath.
The primary effect on droplet formation by the electrostatic 7.1 Homogeneity of Beads:
potential is to direct charged molecules to the surface of the 7.1.1 It has been shown that the properties of the gel
droplet to counteract surface tension (11). Using this type of strongly depend upon the method of preparation. When a gel
instrument, beads below 200 µm and with a small size bead is formed by diffusion of calcium ions into droplets of
distribution may be generated. The desired bead size is alginate solution, a non-uniform distribution of polymer in the
obtained simply by adjusting the voltage (electrostatic poten- bead is obtained. This can be explained by differences in the
tial) of the instrument. The principle for making smaller beads diffusion rate of the gelling ions into the bead relative to the
by electrostatic potential bead generators is shown in Fig. 1. diffusion rate of alginate molecules towards the gelling zone
6.2.4 Vibrating Capillary Jet Breakage—Avibrating nozzle (30).
generates drops from a pressurized vessel. 7.1.2 Another factor that affects homogeneity is the pres-
+ 2+
6.2.5 Rotating Capillary Jet Breakage—Bead generation is ence of non-gelling ions like Na or Mg . Such ions will
achieved by cutting a solid jet of fluid coming out of a nozzle compete with the gelling ions during the gelling process,
by means of a rotating cutting device. The fluid is cut into resulting in more homogeneous bead. More homogeneous
cylindrical segments that then form beads due to surface beads will also be mechanically stronger and have a higher
tension while falling into a gelling bath. porositythanmoreinhomogeneousbeads.Forexample,adding
6.2.6 Emulsification Methods. sodium chloride together with calcium chloride results in the
6.3 Type of Solvent (that is, Cell Growth Medium or formation of a more homogeneous gel bead. Maximum homo-
Water)—The conformation of the alginate molecule will vary geneityisreachedwithahighmolecularweightalginategelled
with changes in the ionic strength of the solute. Therefore, the with high concentrations of both gelling and non-gelling ions.
FIG. 1 Principle of Electrostatic (left) and Coaxial Air Flow (right) Bead Generators
F2315–03
7.2 Gel Porosity and Diffusion: preferred in biomedical applications be
...