Project Page - Cell laden porous gel for liver tissue engineering

Pitfalls : Cell viabilty decrease due to sucrose: osmotic shock kills cells, gelling time is important

Alternatives : change porogen from sucrose to alginate or other dissolvable materials(gelatin)

PAPER TITLE : Liver tissue engineering with cell laden porous gel

A) Background

In tissue engineering application, scaffold is an essential component for highly structured three dimensional tissue architectures, providing a substrate of cell attachment, growth and proliferation. Scaffold materials are traditionally biodegradable synthetic or natural materials, for instance, poly lactic acid(PLA) and polyglycolic acid (PGA) and their mixtures, poly ethylene glycol and poly e-carprolacton as synthetic polymeric materials and collagen, gelatin, fibrin, hyaluronic acid, alginate, chitosan and agarose as natural origin materials [1]. Among them, hydrogel materials are used as promising scaffold materials for a cell laden hydrogel and patterning [2, 3]. Hydrogels are frequently used as base materials in immune-protective microencapsulation of cells for the application of bioreactors and for implantable organ units, as well as for the tissue engineering of cartilage, blood vessels. They exhibit high diffusive permeability to oxygen, nutrients, and other water-soluble metabolites [4]. Hydrogel based scaffolds have advantages in maintaining metabolic activity by high diffusion rate of soluble chemicals and metabolites in the bulk of 3D cell cultures [5]. Pores in tissue engineering scaffolds are introduced not only to enhance the diffusion of metabolic elements but also provide space for cell survival and proliferation and ultimately form 3D interconnected tissue formation [6].

Until now, pore generation in hydrogel scaffolds was not significantly different from that of conventional dense polymeric material based scaffolds. To mention a few of them, salt leaching [7, 8], phase separation and freeze drying (eg. oil emulsion method)[9], gas forming, laser ablation and 3D stereolithographic printing[6, 10] are generally adopted for pore formation in hydrogel scaffold. However, pore generation in cell laden hydrogel scaffolds showed limited success because there are small window of choice in chemical and physical process conditions for maintaining cell viability and desired physical and biological functionality. Inspite of these limitations, cell laden hydrogel is an attractive approach in tissue engineering, because this concept enhances the feasibility of 3D cell patterning [11] by making building block of 3D tissue constructs in addition to the advantages of current hydrogel scaffolds. Furthermore, homogeneous and controllable cell density throughout the scaffolds is desirable in cell seeding efficiency and proliferation of cells throughout the scaffolds. These advantages can overcome the problems of conventional scaffold surface cell seeding methods, which has trouble in cell ingrowth and eventually inhomogeneous tissue formation. In addition to cell homogeneity, pores in the cell laden hydrogel can provide higher diffusion rates of nutrients and space for cell proliferation and reside.

Alginate is a natural origin polysccharide [proof], which is extracted from seaweeds [ref]. This material shows high cytocompatibility and has been used for a long time as a matrix hydrogel for cell culture [ref]. It is widely used in combination with cell technology, such as encapsulation of liver, pancreatic cells and embryonic stem cells for immunoisolation [ref], and printing cells in 3D constructs for elaborated tissue engineering [ref]. Alginate is advantageous in gelling process by contact with calcium ion in aqueous solution, and forms a reversible crosslink. The hydrogels are easily dissolved with calcium chelating solutions, which can be applied in tissue engineering for programmable dissolution of the scaffold matrix material [ref].

Gelatin is a fragmented protein from extracellular collagen molecules. This is thermoresponsible material, gelation under low temperature and swells and dissolves in high temperatures. Under low temperature, gelatin gellates by forming hydrogen bond among the functional groups of the protein backbone [ref]. This hydrogel-liquid phase transformation process is reversible by temperature change, if not crosslinked [ref].

In this paper, we suggest porous cell laden hydrogel by introducing microporous structure in alginate hydrogel matrix. Theremo-responsible gelatin microspheres were used for pore generation in the alginate hydrogel. By combining two different kind of gelling mechanism can provide non cytotoxic environment for cell laden hydrogels resulting highly controllable pore structures. The feasibility of liver tissue engineering application was evaluated with this alginate-gelatin microbead cell laden porous hydrogel scaffold. To evaluate the enhanced diffusion rates of nutrients and oxygen, liver cell line HepG2 was used because this cell line shows high metabolic rate and albumin synthesis during culture. The effect of porosity on cell proliferation and liver cell specific function was evaluated and the characteristics of thermoresponsive hydrogel as porogen in cell laden hydrogel were investigated.

B) Specific Aims

In this study, we suggest porous hydrogel to enhance tissue formation. Alginate hydrogel was used scaffold material for programmable biodegradation. Thermally gelated gelatin beads were used for pore generation in the alginate hydrogel.

Liver cell line HepG2 were used to investigate feasibility of liver tissue engineering for higher architecture fabrication. The effect of pores to cell proliferation, viability and liver cell specific function was assayed with different time scale.

C) General Experimental Approach (Materials and Methods)

Scheme

1. Porogen : sucrose==> gelatin beads

2. Gel material : Alginate mixed with fibrin

3. Porosity : 0, 10, 30, 50%, gel handling : cell strainer, printing film

Gelatin bead preparation

Gelatin type A from porcine skin with bloom 300 (Sigma) were used for pore generating material. Gelatin microbeads were fabricated via water in oil (W/O) method reported previously[ref]. In brief, 5mL of 5% gelatin solution dissolved in DW was autoclaved and pumped to 20 mL mineral oil with 60ml/h flow rate, stirring speed 600 rpm under 40 oC condition. One percent (v/v) of tween 20 were added to avoid aggregation of water phase. After gelatin addition, oil-gelatin solution mixture was stirred 10 minutes more and cooled with ice for 10 minutes for gelation. The resulting beads in oil were mixed with 4oC water and separated by centrifuge 700 rpm for 5 min. Separated gelatin microspheres were collected with sieve and we obtained 150-300 um diameter microspheres. Mineral oil traces were removed by washing the gelatin beads 2 times with DW containing 0.5% tween 20 and 3 times with copious DW. For cell culture, gelatin microspheres were sterilized with 70% ethanol solution for 4 h and resuspended in DW for 2 days. Gelatin microspheres were stored in refrigerator before used. All the DW used here were cooled to 4oC before use.

Porous hydrogel scaffold fabrication

Alginate solution was prepared by dissolving 2g sodium alginate (Sigma **) in 100mL PBS. Gelatin beads were mixed with alginate solution and gelated in agarose mold. Agarose molds were prepared by dissolving 2g agarose in 100mL DW under 80oC and adding 2g CaCl2 before gelation.

Prior to mixing, gelatin beads were collected with 150 um sieve and removed water with gentle vacuum under sieve and then mixed with alginate solution with predetermined ratio. Alginate-gelatin mixture was moved to cell pellet container and mixed again to make homogeneous cell suspension. Cell containing alginate-gelatin microsphere solution was moved to molds and crosslinked alginate molecules with Ca++ ions which diffused from agarose hydrogel molds [fig 1]. These agarose molds were disc shape with 10 mm diameter and 2 mm thickness. After gelation, cell encapsulated alginate-gelatin hydrogel formed and gelatin beads were dissolved in 37 oC.

The resulting porous alginate scaffolds were investigated with SEM after freeze drying, and mechanical stiffness were measured with universal test machine (Instron). Pore size and porosity were measured with mercury intrusion porosimeter.

Morphology of porous alginate hydrogels

The surface morphology of the porous alginate hydrogels was characterized with a Hitachi S-570[proof] scanning electron microscope (SEM) operated at 12kV. The freeze-dried alginate samples were sputter-coated with gold (~20 nm) prior to the morphological examination. The pore density of the porous alginate hydrogels was measured using an image analysis tool (Image J) and was calculated using equation(Naguib, H. E.; Park, C. B.; Panzer, U.; Reichelt, N. Polym Eng Sci 2002, 42, 1481-1492.)

where N is the number of pores, L is the linear length of the area, and M is a unit conversion resulting in the number of the cells per cm3.

Permeability evaluation of porous alginate hydrogel

Water permeability in alginate hydrogel was evaluated with different initial gelatin microsphere content. Porous alginate discs were inserted in the middle of holder and sealed by two o-rings. Both sides of specimens were filled with water with initial height difference of 100cm H2O as shown in fig 2. The water height was recorded with 5 min interval. The measured water front velocity was recorded and compared with control non porous alginate hydrogel.(Li S, de Wijn JR, Li J, Layrolle P, de Groot K. Macroporous Biphasic Calcium Phosphate Scaffold with High Permeability/Porosity Ratio. Tissue Engineering 2003;9(3):535-548.)

Diffusion property measurement of porous hydrogel

To evaluate the transport of nutrients in the porous hydrogel scaffolds, FITC-Dextran with molecular weight 70kDa was used for diffusion measurement. Diffusion evaluation, glass pipettes with inner diameter 1mm were filled with 100 microliter of alginate-glatin microsphere mixtures and gelated with 1% CaCl2 solution for 10 min. Gelatin microspheres were dissolved by increasing temperature and washed in water for 30min. One percent of FITC solution was filled in one side of pipette and sealed with parafilm. Fluorescence of pipettes were obseved with fluorescence microscope and fluorescence picture with 5 min interval. Fluorescence levels of collected images were analyzed with Image J software and diffusivity was estimated.

Cell culture and liver cell function assay

Liver cell line HepG2 cells were added to alginate-gelatin bead mixture before gelation in agarose molds. Gelatin microsphere mixing volume was adjusted to 0, 25, 50, 90% with respect to final alginate-gelatin mixture solution volume and final cell concentration was adjusted to 5 x 10^6 cells/ml. After gelation, each cell encapsulated alginate-gelatin disc samples were moved to 24-well plates and added 2 mL of Dulbecco's Modified Essential Medium(DMEM) containing 10% fetal bovine serum(FBS) and 1% streptomycin, pennicilin. After 2 h, culture medium was changed to remove dissolved gelatin. Cell culture medium was changed 2-day interval.

Biocopatibility of alginate hydrogel

To investigate the efffect of the cell adhesion on the surface of the hydrogel scaffold, alginate hydrogel scaffolds were gelated with various content of fibrin gel. Fibrinogen was dissolved in PBS with 1 wt % concentration under 37oC and filtered, cool down to 4oC. For gelation, fibrinogen, thrombin and alginate solution were mixed together, bubbles were removed with vacuum for 30 sec and poured to agarose molds.

Fibrinogen and alginate solutions were mixed with different ratio of 0, 25, 50, 75, 100% fibrinongen content. Final thrombin mixing ratio was matained to 5 IU/mL. Alginate gelation process was kept 4oC and the samples were moved to 37oC incubator for 20 minutes to fibrin gel formation.

Evaluation of Cell function in porous hydrogel

Cell laden alginate hydrogel samples were used for cell viability, proliferation and albumin assay. For cell viability Live/Dead cell viability kit(Invitrogen-proof) was used. For live/dead cell viability evaluation, each samples were washed with DPBS and assay solution was added. Assay solution was Dulbecco's modified phosphate buffered saline (DPBS) mixed with 0.5 uL calcein-AM and 2.0uL Ethidium homodimer(Eth-D) per 1mL and incubated room temperature for 20 minutes.

Cell proliferation was confirmed by two methods, mitochondrial activity assay with WST-1 (Roche) and direct count with hemocytometer after dissolving the alginate scaffolds with EDTA solution [ref-SNU].

Albumin produced by liver cells werer assayed with ELISA kit (Bethyl-proof) according to the user guide. In brief, each cell cultured medium was collected and centrifuged for assay. And enzyme-linked substrated microwell plates were sequentially prepared by precoating, enzyme link, post coating and fluorescence dye linking. Before change to each step, all the wells were washed with washing buffer. The final solutions were assayed with ELISA reader [proof] and compared with the result of standard solution.

SEM samples were prepared by fixation with 2% glutaraldehyde solution for 1h, 1% osmium tetraoxide for 1h and dehydration with serial ethanol solution and freeze drying.

Immunofluorescence and histological staining

Actin filaments were immunolabelled with Alexa 596 phalloidin(Invitrogen). In brief, all the samples were washed with DPBS for 5 min, and fixed with 4% paraformaldehyde solution for 30 min, permeabilized by 1 % triton X-100 in DPBS solution, blocked with 3% BSA, and immunolabelled with Alexa 596 phalloidin in 1:40 dilution. After each step, all the samples were washed with DPBS 3 times for 3minutes. After staining the actin filaments, samples were coated with DAPI containing fixation gel [Name proof needed-freezer].

D) RESULTS AND DISCUSSION (Design Pitfalls and alternatives)

Expected results

1. Cell number, spheroid number(day 14) increase with porosity, culture periods

2. Cell viability increase with porosity, culture periods

3. Albumin secretion increase with porosity, culture periods

Things to mention in results and discussion (and Figure)

- Cell laden hydrogel microstructure (SEM): with/without cell(none, day 1, day 9)

- Cell viability in porous hydrogel

- Cell proliferation (WST-1 and number count)

- Albumin secretion in alginate hydrogel

- Liver cell spheroids number in hydrogel : SEM and EDTA dissolution

E) Potential Figures

Samples : 2% alginate, gelatin 0, 25%, 50%, 80%

Cell concentration : 5x10^6 cells/mL

Mechanical test : 5 samples each condition

SEM : 2 samples without cells, 2 samples with cells

Figure 1 : Schematic of experiment, Microstructure of porous hydrogel (SEM)

Figure 2 : Porosity, mechanical properties of hydrogel : Compression vs porogen conten

Figure 3 : Cell viability with porogen content, time ( day = 1, 3 ,5, 7, 9)

Alginate-gelatin gelated-HepG2, cultured 1 day

Figure 4 : Cell proliferation with porogen content, time( day = 1, 3 ,5, 7, 9)

Figure 5 : Cell morphology actin staining, SEM (day 1, 3, 5, 7, 9)

Figure 6 : Albumin secretion with time( day = 1, 3 ,5, 7, 9, 11)

Figure 7. Albumin staining (day = 1, 3, 5, 7, 9)

Figure 8. CYP 450 activity with culture time (day = 1, 3, 5, 7, 9)

F) Future Directions

Cell adhesive properties of matrix hydrogel can affect the results. : try later with changing the composition of hydrogel materials with fibrin or gelatin

Cell laden alginate(gelatin) microsphere application to agarose(alginate) hydrogel : ESC culture