ASTM F2315-18

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Designation: F2315 − 11 F2315 − 18
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
1.1 This guide discusses information relevant to the immobilization or encapsulation of living cells or tissue in alginate gels.
Immobilized or encapsulated cells are suitable for use in biomedical and pharmaceutical applications, or both, including, but not
limited to, Tissue Engineered Medical Products (TEMPs).
1.2 This guide addresses key parameters relevant for successful immobilization and encapsulation in alginate gels.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory requirementslimitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F1251 Terminology Relating to Polymeric Biomaterials in Medical and Surgical Devices (Withdrawn 2012)
F1903 Practice for Testing for Cellular Responses to Particles in vitro
F1904 Practice for Testing the Biological Responses to Particles in vivo
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devicesand is the direct responsibility of Subcommittee F04.43
on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved March 1, 2011Nov. 1, 2018. Published March 2011December 2018. Originally approved in 2003. Last previous edition approved in 20102011
as F2315 – 10.F2315 – 11. DOI: 10.1520/F2315-11.10.1520/F2315-18.
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 − 18
F1905 Practice For Selecting Tests for Determining the Propensity of Materials to Cause Immunotoxicity (Withdrawn 2011)
F1906 Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte Proliferation,
and Cell Migration (Withdrawn 2011)
F2064 Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and Tissue
Engineered Medical Product Applications
F2312 Terminology Relating to Tissue Engineered Medical Products
2.2 USP Document:
USP Monograph USP 24/NF1940/NF35 Sodium Alginate
2.3 Other Referenced Documents:
EN-ISOISO 10993 Biological Evaluation of Medical DevicesDevices—Part 1: Evaluation and Testing Within a Risk
Management Process
International Conference on Harmonization (ICH) S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of
Pharmaceuticals (July 1997)
21 CFR Part 312 Code of Federal Regulations Title 21, Part 312 Investigational New Drug Application
3. Terminology
3.1 Definitions:
3.1.1 alginate, n—polysaccharide obtained from some of the more common species of marine algae, consisting of an insoluble
mix of calcium, magnesium, sodium, and potassium salts. F2312
3.1.1.1 Discussion—
Alginate exists in brown algae as its most abundant polysaccharide, mainly occurring in the cell walls and intercellular spaces of
brown seaweed and kelp. Alginate’s main function is to contribute to the strength and flexibility of the seaweed plant. Alginate
is classified as a hydrocolloid. The most commonly used alginate is sodium alginate. Sodium alginate and, in particular, calcium
cross-linked alginate gels are used in Tissue Engineered Medical Products (TEMPs) as biomedical matrices, controlled drug
delivery systems, and for immobilizing living cells.
3.1.2 APA bead, n—alginate-poly-L-lysine-alginate bead. F2312
3.1.3 encapsulation, n—a procedure by which biological materials, such as cells, tissues, or proteins, are enclosed within a
microscopic or macroscopic semipermeable barrier. F2312
3.1.4 endotoxin, n—pyrogenic high molar mass lipopolysaccharide (LPS) complex associated with the cell wall of
gram-negative bacteria. F2312
3.1.4.1 Discussion—
Though endotoxins are pyrogens, not all pyrogens are endotoxins. Endotoxins are specifically detected through a Limulus
Amebocyte Lysate (LAL) test.
3.1.5 gel, n—the three-dimensional network structure arising from intermolecular polymer chain interactions. Such chain
interactions may be covalent, ionic, hydrogen bond, or hydrophobic in nature. See also Terminology F1251.
3.1.6 immobilization, n—the entrapment of materials, such as cells, tissues, or proteins within, or bound to, a matrix.
3.1.7 pyrogen, n—any substance that produces fever. F2312
3.2 Additional definitions regarding alginate may be found in Guide F2064. Additional definitions regarding polymeric
biomaterials may be found in Terminology F1251F2312.
4. Significance and Use
4.1 The main use is to immobilize, support, or suspend living cells or tissue in a matrix. The use of an encapsulation/
immobilization system may protect cells or tissues from immune rejection. When immobilizing biological material in alginate gels,
there are numerous parameters that must be controlled. This guide contains a list of these parameters and describes the methods
The last approved version of this historical standard is referenced on www.astm.org.
Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758, 1211 Geneva 13, Switzerland.https://www.fda.gov/downloads/drugs/
guidancecomplianceregulatoryinformation/guidances/ucm074929.pdf or http://www.ich.org/products/guidelines/safety/safety-single/article/guidance-on-genotoxicity-
testing-and-data-interpretation-for-pharmaceuticals-intended-for-human-use.html
Available from U.S. Government Printing Office, Superintendent of Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http://www.access.gpo.gov.
F2315 − 18
and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of an encapsulation
system using alginate. This guide only covers single gelled beads, coated or not, and not double capsules or other constructs.
4.2 The alginate gelation technology covered by this guide may allow the formulation of cells and tissues into biomedical
devices for use as tissue engineered medical products or drug delivery devices. These products may be appropriate for implantation
based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a
guarantee of clinical success in any tissue engineered medical product or drug delivery application.
5. Gelation Techniques
5.1 Most methods for encapsulation of cells or tissue in alginate gels (see USP Monograph USP 40/NF35) basically involve two
main steps. The first step is the formation of an internal phase where the alginate solution containing biological materials is
dispersed into small droplets. In the second step, droplets are solidified by gelling or forming a membrane at the droplet surface.
5.2 The most simple and common way to produce small beads or capsules is by forming droplets of a solution of sodium
alginate containing the desired biological material (cells, tissues, or other macromolecules) and then exposing them to a gelling
2+ 2+ 2+
bath. A gelling bath may be a solution containing divalent cross-linking cations such as Ca , Sr , or Ba . Monovalent cations
2+ 8
and Mg ions do not induce gelation of alginates (1).
5.3 Concentration of Ions:
5.3.1 The concentration of gelling ions used must be determined based upon factors such as desired gel strength, type of alginate
used (G- or M-rich), M-rich (see X2.1), and isotonicity of the gelling solutions. Calcium ion concentrations of from 50 mmol/L
to 150 mmmmol/L are often used.
2+ 2+ 2+
5.3.2 Other gelling ions may be used, such ions, (such as Ba or Sr . ) may be used. The concentration of Ba in the gelling
solution must be determined based upon the desired characteristics of the final gel and on regulatory and toxicological
2+
considerations as Ba can induce toxic effects in cells.
5.3.3 Concentration of Non-gelling Ions—Various additives present in the gelling solution that do not participate in the
+
formation of cross-links constitute non-gelling ions. These ions may be Na , which can be used to produce homogeneous gels (see
7.1), ions present in cell culture medium (if present in the gelling bath), and others.
6. Formation of Beads
6.1 Bead size is one of the most important parameters of alginate gel beads and capsules in biomedical applications. The
appropriate size will often be a compromise. The bead itself must be large enough to contain the biological material. Larger beads
are also easier to handle during washing or other treatments. In many applications involving cells, the cells should be
homogeneously distributed within the internal capsular matrix. When generating beads, the desired mean size and acceptable size
distribution should be accounted for. The size of the beads is primarily controlled by regulating droplet formation.
6.2 Droplet Size—Droplet size is dependent upon several factors: The size of the material to be immobilized or encapsulated
(that is, single cells or cell aggregates such as pancreatic islets), the technique used to generate droplets (that is, pipette or syringe,
coaxial air flow, electrostatic generator, jet-cutter,jet-cutter and so forth) and the viscosity of the alginate solution. Generally, for
biomedical applications, droplet size is regulated to give a gelled bead having a diameter of <200 to 1000 μm. Per unit volume,
smaller beads yield a larger surface area to transplant area-to-transplant volume, a ratio that results in enhanced survival of tissue
due to better nutritional and oxygen supply. Various techniques can be used to form droplets as described in more detail by Dulieu
et al. (2). These include, but are not limited to:
6.2.1 Extrusion through a Needle—Beads can be made by dripping an alginate solution from a syringe with appropriate diameter
needle directly into a gelling bath. While this method does not require any instrumentation, the size and size distribution of the
produced beads are difficult to control.
6.2.2 Coaxial Air or Liquid Flow—The coaxial air jet system is a simple way of generating small beads (down to around 400
μm), although the size distribution will normally be larger as compared to an electrostatic system. In this system, a coaxial air
stream is used to pull droplets from a needle tip into a gelling bath (Fig. 1).
6.2.3 Electrostatic Potential—An electrostatic potential can be used to pull droplets from a needle tip into a gelling bath. The
primary effect on droplet formation by the electrostatic potential is to direct charged molecules to the surface of the droplet to
counteract surface tension (2). Using this type of instrument, beads below 200 μm in diameter and with a small size distribution
may be generated. The desired bead size is obtained simply by adjusting the voltage (electrostatic potential) of the instrument. The
principle for making smaller beads by electrostatic potential bead generators is shown in Fig. 1.
6.2.4 Vibrating Capillary Jet Breakage—A vibrating nozzle generates drops from a pressurized vessel.
6.2.5 Rotating Capillary Jet Breakage—Bead generation is achieved by cutting a solid jet of fluid coming out of a nozzle by
means of a rotating cutting device. The fluid is cut into cylindrical segments that then form beads due to surface tension while
falling into a gelling bath.
6.2.6 Emulsification Methods.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
F2315 − 18
FIG. 1 Principle of Electrostatic (left) and Coaxial Air Flow (right) Bead Generators
6.3 Type of Solvent (that is, Cell Growth Medium or Water)—The conformation of the alginate molecule will vary with changes
in the ionic strength of the solute. Therefore, the apparent viscosity of an alginate solution may change, depending upon whether
the alginate is dissolved in water or in a salt-containing medium. When using droplet generators, size and sphericity of the beads
will, therefore, depend on the viscosity of the alginate solution and the distance the droplets fall before reaching the gelling
solution. In addition, the final size of the beads will be dependent ofon the gelling conditions used.
6.4 Concentration of Biological Material (Cells or Others)—In applications involving immobilization of cells diffusion
properties of different molecules within the beads will also depend strongly on the load of cells. As a consequence of diffusion
limitations cells surrounded by other cells within the gel network may, therefore, be strongly influenced by the metabolism of the
surrounding cells. As a result surrounded cells may be trapped in a micro-environment lacking essential nutrients like oxygen. This
may typically result in cell death in the center of the beads with an outer rim of viable cells.
6.5 Presence of Impurities—Several authors (3, 4) found that perfectly spherical and smooth alginate beads could only be
formed by using a highly purified alginate.
6.6 3D-Printing-Alginates are being widely used as bioinks for 3D-printing (additive manufacturing) of encapsulated cells for
engineering tissues. (5, 6)
7. Final Capsule or Bead Properties
7.1 Homogeneity of Beads:
7.1.1 It has been shown that the properties of the gel strongly depend upon the method of preparation. When a gel bead is
formed by diffusion of calcium ions into droplets of alginate solution, a non-uniform distribution of polymer in the bead is
obtained. This can be explained by differences in the diffusion rate of the gelling ions into the bead relative to the diffusion rate
of alginate molecules towards the gelling zone (57).
+ 2+
7.1.2 Another factor that affects homogeneity is the presence of non-gelling ions like Na or Mg . Such ions will compete with
the gelling ions during the gelling process, resulting in more homogeneous bead.beads. More homogeneous beads will also be
mechanically stronger and have a higher porosity than more inhomogeneous beads. For example, adding sodium chloride together
with calcium chloride results in the formation of a more homogeneous gel bead. Maximum homogeneity is may be reached with
a high molecular weight high-molecular-weight alginate gelled with high concentrations of both gelling and non-gelling ions.
7.2 Gel Porosity and Diffusion:
7.2.1 For many applications, particularly when capsules are used to limit or restrict certain solutes, for example antibodies for
rejection, knowledge about the diffusion characteristics, pore size and pore size distribution is important. Electron microscopy and
inverse size exclusion chromatography hashave been used to study porosity of alginate gels (68, 79, 810). It has been found that
pore size may range in size from 5 to 200 nm in diameter (911).
7.2.2 Diffusion of large molecules, such as proteins, requires a more open pore structure. Therefore, the gel network may restrict
the transport of larger molecules. The highest diffusion rate of proteins, indicating the most open pore structures, is found when
gels are made using high G alginates (see X2.1) (1012, 79). Diffusion coefficients also increase when lowering the alginate
concentration.
7.2.3 Protein diffusion is faster for homogeneous beads than for inhomogeneous beads where the alginate is concentrated at the
surface (79).
7.2.4 Porosity of an alginate bead may also be reduced by partially drying. Beads made with a high G-content alginate (see
X2.1) will swell only slightly when returned to water, and the resulting increased alginate concentration will reduce the average
pore size.
F2315 − 18
7.2.5 The gel network to a lesser extent influences diffusion properties of smaller molecules. Diffusion coefficients of molecules
such as glucose and ethanol are typically as high as about 90 % of the diffusion coefficient in water. Tanaka et al. (1113) found
no reduction in diffusion coefficients for solutes with Mw moleular weight < 2 × 10 in calcium alginate gel beads as compared
with free diffusion in water.
7.2.6 Diffusion within the gel network is not solely dependent upon porosity. Since the gel matrix is negatively charged,
electrostatic forces between the gel network and ionic substrates should also be considered (1214). For example, the rate for BSA
(bovine serum albumin) diffusion out of alginate beads increased with increasing pH (79), presumably due to the negative charge
on the protein as the pH increased. The negative chargedcharge of the alginate matrix is also responsible for a difference between
influx and efflux of molecules. At pH 7, most proteins are negatively charged and will therefore not easily diffuse into the matrix.
When such proteins are immobilized in a gel, the repulsive forces result in an efflux that is greater than their free molecular
diffusion rate (1315).
7.2.7 There may be other contributing factors to diffusion of molecules through, into, or out of, the gel. Diffusion of drugs, or
other molecules of interest, or the molecular weight cut-off of the gel network itself, needs to be experimentally determined, if
important for the functionality of the TEMP.
7.3 Gel Strength and Stability:
7.3.1 Mechanical properties of alginate beads will to a large extent vary with the alginate composition (1012). The highest
mechanical strength is found when the G-content is more than about 70 % and average length of G blocks (NG > 1) of about 15.
For molecular weights above a certain value, the mechanical strength is determined mainly by chemical composition and block
structure, and is therefore independent of the molecular weight. However, low molecular weight alginates are often preferred in
biomedical applications because they are easier to sterilize by membrane filtration. Below a certain critical molecular weight, the
gel forming ability is reduced. This effect will also be dependent of the alginate concentration because of polymer coil overlap.
7.3.2 The alginate gel as an immobilization matrix is sensitive to chelating compounds such as phosphate, lactate and citrate,
+ 2+
lactate, citrate and ethylenediaminetetraacetic acid (EDTA), and the presence of anti-gelling cations such as Na or Mg . To avoid
+
this, gel beads may be kept in a medium containing a few millimolar millimoles of free calcium ions and by keeping the Na :
2+ 2+
Ca ratio less than 25:1 for high G alginates and 3:1 for low G alginates (1012). An alternative is also to replace Ca with other
divalent cations with a higher affinity for alginate. There has been found a correlation between mechanical gel strength and affinity
2+ 2+ 2+ 2+ 2+ 2+
for cations (810). It was found that gel strength decreased in the following orders: Pb > Cu = Ba > Sr > Cd > Ca >
2+ 2+ 2+ 2+ 2+ 2+
Zn > Co > Ni . However, in applications involving immobilization of living cells only Sr , Ba , and Ca are considered
non-toxic enough for these purposes (1315).
7.4 Coating of Alginate Gel Beads:
7.4.1 As alginates may form strong complexes with polycations such as chitosan or polypeptides, or synthetic polymers such
as polyethylenimine they may be used to stabilize the gel. When used as coating materials, such complexes may also be used to
reduce the porosity. Alginate gels have been found to be stable in a r
...