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

Summary

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.

Abstract

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.

Introduction

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.

Spe....

Protocol

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....

Results

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 (i........

Discussion

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 .......

Materials

List of materials used in this article

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