Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Podsumowanie

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Streszczenie

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.^2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.⁴ As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.^2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the “tissue”. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

Wprowadzenie

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.^5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.^13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.^18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types.

Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel²² for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.^1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

Protokół

1. Creation of pNIPAAM-coated Plates

1. Dissolve the 2.6 g of pNIPAAM in 2 ml of a 60% toluene/40% hexane solution.
2. Heat the mixture to 60 °C for 10 min stirred, until the pNIPAAM is dissolved.
3. Cut filter paper into a 60 mm diameter circle and place paper in the Buchner funnel.
4. Filter the solution through Buchner Funnel into the pre-weighed glass beaker (do not use plastics, as hexane will melt plastics).
5. Place the beaker and contents into a bell vacuum (24 psi) O/N (16 hr). Note: Until the residue is reacted with isopropyl it will oxidize so make sure it does not come into contact with oxygen.
6. Weigh the beaker to establish the weight of the pNIPAAM.
7. Add isopropyl alcohol to the pNIPAAM, creating a 50/50 w/w solution.
8. Place 2 ml of the solution on the surface of the tissue culture plate, and coat for 5 min under UV light.
9. Wash the plate with 2 ml of warm PBS twice before using for cell culture.

2. Creation of Cell Sheets

Note: Cell sheets of primary cells for the target organ can be created using a number of different methods, or by coating tissue culture surfaces with thermo-responsive polymer as described here. Pre-coated thermo-sensitive plates are also offered by a number of vendors.

Note: This protocol is for culture using a 35 mm dish. Briefly, cells are first incubated at 37 °C for a minimum of 24 hr at confluence to establish lateral connections between adjacent cells. To release cell sheets, plates are subjected to temperatures below 32 °C. The cell sheet is then transferred to the strong base fibrous matrix containing a neovascular permissive hydrogel with vascular endothelial cells.

1. Isolate the cell population. Note: This method is dependent on the individual derivation procedures and the type of cells. Rat aortic smooth muscle cells (RASMC) are used in this example. These are primary smooth muscle cells isolated from the abdominal aorta of a rat.
2. Wash the cells with 2 ml of warm PBS.
3. Add 3 ml of trypsin (or other cleaving/disassociating solution) to the cells for 5 min.
4. Inhibit the trypsin by the addition of 3 ml of the culture media, or phosphate buffer solution (PBS) containing 10% Fetal Bovine Serum (FBS).
5. Collect the cells in a conical tube and count an aliquot.
6. Spin the cells at 1,000 rpm (228 x g) for 5 min.
7. Aspirate the supernatant and resuspend the cells in their growth media (SmGM2 plus bullet kit culture medium is used for RASMC).
8. Place the media containing the cells on a 35 mm thermo-sensitive plate – pNIPAAM coated plate at a concentration that will achieve 100% confluence. Note: For RASMCs that number was determined to be 100,000 cells/cm². However, due to loss of cells during the passing, 120% of that value is used.
9. Place into an incubator at 37 °C O/N. Note: It is important to maintain the cells at 37 °C to maintain the cell adhesion to the plate.

3. Preparation of Foundational Matrix

Note: Various 3D fibrous matrices can be used to layer strong fibrous matrix between the delicate cell sheets. Some examples include: gelfoam, bioglass, natural acellularized materials²⁶ or nanospun materials^27,28 The porcine urinary bladder matrix (UBM) used in these studies was generously provided from our collaborator, Dr Badylak.²⁹

1. Prior to use, determine matrix characteristics including the lack of cellular content if decellularized matrix is used,^27,28 cell specific viability, and void space.²²
2. Cut the pre-sterilized matrix into a desired size and shape. Note: Here, a hole-punch is used to cut a 4 mm diameter circle.

4. Seeding Endothelial Cells into a Neovascular Permissive Hydrogel

Note: Endothelial cells can be obtained from a variety of sources, including differentiation from stem or progenitor cells. Here, HuVECs are used.

1. Use any permissive hydrogel (fibrin, collagen gels) as long as the cross-linking time is short enough to allow the cells stay viable. Note: Here, a Hyaluronan (HA) based gel cross-linked with a disulfide bridge is used.
2. Prepare the HA hydrogel in accordance with the company protocol.
3. Collect the endothelial cells and disperse into a single cell solution using 1x trypsin. Note: Accutase or Cell Dissociation Buffer could also be used for single cell dispersion.
4. Deactivate the trypsin enzyme by using an equal amount of soybean trypsin inhibitor (if it is important that cells do not come into contact with serum) or 10% FBS in PBS, collecting the solution/cells into a 15 ml conical tube.
5. Count the cells, and calculate the volume needed for the patch dimensions (previously quantified). Note: For a 4 mm patch, here 2 million endothelial cells are used.
6. Extract 2 million cells, and place into a new 15 ml conical tube.
7. Spin at (228 x g) for 5 min.
8. Aspirate the supernatant, leaving the cells as a pellet in a conical tube.
9. Mix the HA and Gelatin liquid materials in a 1:1 ration. Then add 80% of the total volume into the conical tube containing the pellet.
10. Resuspend the endothelial cells in the 1:1 HA /Gelatin mixture
11. Place the suspended cells in the HA /Gelatin mixture into the base fibrous matrix from Step 2.
12. Add 1/5 (20 percent) of the total volume desired of the cross-linker
13. Incubate for 1 hr at 37 °C.

5. Isolation of Cell Sheets

1. Remove the 35 mm pNIPAAM-treated plates containing the cells from the incubator and place in a cell culture hood at RT.
2. Quickly aspirate the media from the cells, and add 2 ml of 6% normal gelatin that has been heated to 37 °C.
3. While the gelatin is still warm, place the metal lattice into the gelatin, submerging it below the surface of the normal gelatin (Movie 1).
4. Place the entire plate onto ice for 5 to 7 min, allowing the gelatin to harden.
5. After 7 min, use a spatula to carefully separate the gelatin edges from the side of the plate, and then use forceps to lift the metal lattice from the plate Note: The 6% gelatin, and the cell sheet should lift with the lattice.
6. Move the cell sheet to the dish and place on top of the base fibrous matrix-hydrogel combination, carefully setting the lattice on top of the construct. Note: The apical side of the cell sheet will still be in the top position.
7. Add 2 ml of warm media (37 °C).
8. Incubate O/N allowing the sheet of cells to adhere to the hydrogel surface.
9. Remove the metal lattice after the solution warms (approximately 1 hr), or the next day.

Wyniki

The flow diagram (Figure 1) shows the overall method of making the multilayered patch. Cell sheets are detached from the pNIPAAM treated plate by dropping the temperature below 32 °C. Then the cell sheet is placed on top of the cross-linked hydrogel containing the endothelial cells seeded into the underlying fibrous matrix (Figure 1). The pretreated thermo-sensitive plates can also be used for creating the cell sheets. Special topological surfaces are used to specifically pattern (<...

Dyskusje

The critical steps in the protocol include: coating the plate surfaces with the thermoresponsive polymer and manipulating the cell sheets after cooling the plates. Because different cells exhibit different physical properties, like adhesivity, the lifting time should be optimized for each different cell type. The second, and most significantly challenging component of this protocol, centers on the manipulation of the cell sheet, a critical aspect of methods for tissue assembly. The single cell layer in the cell sheet is ...

Materiały

Name	Company	Catalog Number	Comments
Reagents
Calcein-AM	Invitrogen	C3099	Cell tracker / live dye
Lysotracker Red	Invitrogen	L7528	Cell tracker
Neutral Red	Sigma	N7005	Visible Cell dye
pNIPAAM	Sigma Aldrich	412780250	Poly(N-isopropylacrylamide)
Toluene	Sigma Aldrich	244511-1L
Hexane	Sigma Aldrich	296090-1L
RAOSMC	Lonza	R-ASM-580	Rat Aortic Smooth Muscle Cells
SmGM2	Lonza	CC-4149	Smooth Muscle Media
HUVEC	Invitrogen	C-003-5C	Human Venous Endothelial Cells
HyStem	Glycosan/Biotime
Isopropyl alcohol	VWR International	BDH1133-4LP
Trypsin	Corning Cellgro	25-053-C1
PBS	Gibco	14287-072
FBS	Gibco	16140-071
Specific Equipment
Filter paper	Ahlstrom	6310-0900
Buchner Funnel	Sigma Aldrich	Z247308
UpCell Plates	Nunc	2014-11
UV light	Jelight Company	UVO Cleaner Model No.42