Therese Andersen

Chapter 9:Alginates as biomaterials in tissue engineering

Alginates comprise a rather broad family of polysaccharides found in brown seaweeds (Laminaria sp., Macrocystis sp., Lessonia sp. and others), from which they are produced industrially. The annual production is estimated to approximately 38.000 tons worldwide.1 In addition, a variety of different al...

3D Cell Culture in Alginate Hydrogels

This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

Ionically Gelled Alginate Foams: Physical Properties Controlled by Operational and Macromolecular Parameters

Alginates in the format of scaffolds provide important functions as materials for cell encapsulation, drug delivery, tissue engineering and wound healing among others. The method for preparation of alginate-based foams presented here is based on homogeneous, ionotropic gelation of aerated alginate solutions, followed by air drying. The method allows higher flexibility and better control of the pore structure, hydration properties and mechanical integrity compared to foams prepared by other techniques. The main variables for tailoring hydrogel properties include operational parameters such as degree of aeration and mixing times and concentration of alginate, as well as macromolecular properties such as the type of alginate (chemical composition and molecular weight distribution). Exposure of foams to γ-irradiation resulted in a dose-dependent (0-30 kGy) reduction in molecular weight of the alginate and a corresponding reduction in tensile strength of the foams.

Ionically gelled alginate foams: Physical properties controlled by type, amount and source of gelling ions

A new and flexible method for preparation of dry macroporous alginate foams with the capability of absorbing physiological solutions has been developed, which may find use within areas such as wound healing, cell culture, drug delivery and tissue engineering. The present study demonstrates how the gelation rate of the alginate and degree of ionic crosslinking can be utilized to control the physical foam properties. The rate of released Ca(2+)/Sr(2+) gelling ions available for interaction with the alginate was influenced by the concentration and physical characteristics of CaCO₃/SrCO₃ particles. The method of preparation of such foams allows, as described herein, tailoring of the pore structure, hydration properties and mechanical integrity in a manner not possible by other techniques.

Abstract 307: Determination of cell surface receptors CD44 and ErbB2 in NovaMatrix-3D - a 3D cell culture system

NovaMatrix-3D is an alginate-based cell culture system comprising an alginate foam matrix and an alginate immobilizing solution. Cells are first suspended in a solution of sodium alginate that is then applied to the foam. As the foam absorbs the cell suspension, gelation occurs in situ as calcium ions are donated from the foam which effectively cross-links the added alginate. A hydrogel forms in the pores of the foam entrapping the cells throughout the foam. Cell immobilization occurs at room temperature and physiological pH. Cell localization and multi-cellular structures formed within the transparent gel can be visualized using confocal microscopy after vital staining of cells within the gel. A unique aspect of using an alginate-based foam matrix for 3D cell growth is that cells and intact multi-cellular structures can be retrieved for further analysis by adding calcium chelating agents such as citrate that dissolve the gel. While the retrieval of intact 3D structures is a simple one-step process, it is of interest to evaluate if imaging of cellular receptors can be performed on cells within the 3D hydrogel. Results show that two cell receptors, CD44 and ErbB2, which are upregulated in SKOV-3 cells, can be detected and imaged on multicellular spheroids within the 3D alginate matrix. First, the receptor status of CD44 and ErbB2 were established for SKOV-3 cells growing as a traditional 2D cell culture using flow cytometry. SKOV-3 cells were then immobilized in NovaMatrix-3D using 150 μM RGD-alginate. The presence of RGD has previously been shown to accelerate cell proliferation and 3D cell growth of SKOV-3. After 12 days of growth, multicellular spheroids formed. The alginate hydrogel disks where then treated with FITC-anti-CD44, Pacific Blue-anti-CD44, or an FITC-conjugated Affibody directed against ErbB2. Hydrogel discs were also treated with relevant isotypic controls. Confocal microscopy was used to detect and image cellular receptors. The results show that the CD44 hyaluronan receptor could be detected and imaged using two different antibodies. Furthermore, another cell receptor, ErbB2, was visualized using an Affibody. Therefore, it may not be necessary to retrieve multi-cellular structures from NovaMatrix-3D in order to visualize and image cellular receptors. Traditionally immobilization of cells within an alginate hydrogel has been used to provide immune-protection to cells used in forming an artificial organ, for example pancreatic islets. However, the alginate hydrogel formed in NovaMatrix-3D is comprised of a lower alginate concentration, has a lower, non-saturating concentration of calcium as a cross-linking ion, and has a more open pore structure. These attributes facilitate influx of antibodies which allows in situ immuno-staining and imaging of cellular receptors. The ability to stain cellular receptors on 3D cell structures within an alginate hydrogel make NovaMatrix-3D an even more versatile cell culture system. Citation Format: John Michael Dornish, Therese Andersen. Determination of cell surface receptors CD44 and ErbB2 in NovaMatrix-3D - a 3D cell culture system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 307. doi:10.1158/1538-7445.AM2015-307

Abstract 3846: The effect of x-ray radiation on spheroids cultured in a 3D matrix under different oxygen conditions .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The use of 3-dimensional (3D) matrices for cell culture has become popular as it provides more representative models of the biological situation in tissues, organs and tumors than the traditional 2D cell culture. NovaMatrix®-3D is an alginate-based cell culture system comprising an alginate foam matrix and an alginate immobilization solution. The cells are first suspended in the alginate solution and applied to the foam. This induces in situ gelation by diffusion of calcium ions to the added alginate and the cells are immobilized inside the pores of the foam. Alginate is not recognized by cell receptors and is considered inert towards cells. Cells form 3-dimensional structures such as spheroids, and the system may be utilized as an in vitro tumor model. The growth of spheroids inside the 3D matrix can be determined by evaluating changes in spheroid volume. Hypoxic cells are more resistant towards traditional cancer treatments such as chemotherapy and radiation. This study used cells cultured in a 3D matrix to evaluate the effect of different doses of x-ray radiation when cells were cultured under different oxygen concentrations. NHIK 3025 cells (human cervical carcinoma) were cultured in 2D and then established as spheroids in NovaMatrix®-3D under 20% O2 and 5% O2 continuous culture conditions. The growth rate of spheroids was first determined without x-ray radiation of the cells. In subsequent experiments the cells were irradiated after 8 days of culture at a dose of 5 Gy or 15 Gy. In all experiments the spheroid growth rate was monitored by determining the volume of the spheroids by vital staining and confocal imaging at regular intervals over 30 days. The volumes were obtained by measuring the diameter of the spheroids in both x- and y-directions. The radii were calculated from the average of these diameters and were then used to calculate the volumes. Spheroids were formed under both normoxic and hypoxic conditions and it was possible to determine growth curves from the volume measurements. The sigmoidal growth curves were similar for both normoxic and hypoxic conditions without radiation treatment. An x-ray radiation dose of 5 Gy was not sufficient to affect the growth rate of the spheroids showing that NHIK 3025 cells are more radiation resistant when grown as 3D spheroids than in traditional 2D culture. When the spheroids were irradiated with 15 Gy, a decrease in growth rate was observed. The spheroids cultured under 20% oxygen were affected more, i.e. the growth curves showed a significant reduction in volume increase, compared to the spheroids cultured under 5% oxygen. This study indicates that hypoxia increases the resistance of NHIK 3025 spheroids against x-ray radiation, and demonstrates the utility of a 3D cell culture matrix (NovaMatrix®-3D) in dose response experiments. This work was funded by the METOXIA project no.222741 under the 7th Research Framework Program of the European Union. Citation Format: John Michael Dornish, Christine Markussen, Therese Andersen. The effect of x-ray radiation on spheroids cultured in a 3D matrix under different oxygen conditions . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3846. doi:10.1158/1538-7445.AM2013-3846

Abstract 5269: Cell matrix interactions in a 3D alginate cell culture system

The use of 3-dimensional (3D) matrices for cell culture is gaining popularity as a substitute for traditional 2D cell culture as it can enable formation of multi-cellular structures and a more biologically relevant cell-cell contact such as found in tissues, organs and tumors. NovaMatrix ® -3D is an alginate-based cell culture system comprising an alginate foam matrix and an alginate immobilizing solution. Cells are entrapped throughout the alginate foam by first suspending them in a solution of sodium alginate that is applied to the foam. As the foam absorbs the suspension, in situ gelation occurs as calcium ions are donated from the foam cross-linking the added alginate forming a hydrogel in the pores of the foam comprising the cells. Cells do not have receptors that recognize alginate molecules and this may stimulate formation of spheroids. However, some cells require signaling molecules and matrix interaction in order to proliferate. This was evaluated by culturing cells in the presence of alginate to which the peptide sequence RGD (Arg-Gly-Asp) was covalently attached (RGD-alginate) thereby facilitating a signaling cascade via integrin-RGD interaction. Various cell lines were first cultured in 2D then prepared as a suspension in 0.5 - 1% alginate or RGD-alginate in cell culture medium. An aliquot of each cell suspension was added to γ-sterilized NovaMatrix ® -3D foams. NHIK 3025 (human cervix carcinoma), OVCAR-3 (human ovarian adenocarcinoma), SKOV-3 (human ovarian carcinoma), NIH:3T3 (murine fibroblasts), C2C12 (murine myoblasts), V79-379A (hamster lung fibroblast) and MDCK (Madin Darby canine kidney) were used and single cells were immobilized in the alginate foams. Cell localization within the foam was visualized using confocal microscopy to identify fluorescently labeled (CellTrace™ CFSE) cells. Cell proliferation was measured by counting cells after de-gelling the foam using sodium citrate. Cell proliferation and spheroid formation occurred during three weeks of culture in foams with or without RGD-alginate for NHIK 3025, SKOV-3, 3T3 and V79-379A indicating that these cell lines do not require the presence of RGD within the alginate matrix for cell growth. However, during the same period, cell proliferation was only seen in the foams containing RGD-alginate for OVCAR-3, MDCK and C2C12. The proliferation rate was affected by the RGD density. Cells that did not proliferate in pure alginate were still metabolically viable as shown by live-dead staining. This cell culture model enables 3D cell culture, formation of spheroids and isolation of intact cellular structures. Cell-matrix interaction can be controlled by type and concentration of signaling molecules covalently attached to the alginate immobilization solution making NovaMatrix ® -3D a versatile cell culture system. Portions of this work have been funded with the support from METOXIA project no.222741 under the 7th Research Framework Programme of the European Union. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5269. doi:1538-7445.AM2012-5269