**5. Bioengineering substitutes that resemble native tissues**

Classical self-assembly technique involves sheet stacking in order to generate a tissue with sufficient mechanical strength. The superimposition of sheets Influences cell distribution. Although fusion of all sheets occurs following sheet stacking, a pattern at the site of each sheet fusion remains visible. Epithelial cell seeding has been noted to reduce sheet demarcations after sheet stacking. Nevertheless, different layers are visible in the 3D self-assembled tissue, Which does not correspond to native stroma architecture and weakens tissue mechanical strength [31, 66, 85, 88]. To outwit this issue, a newly reseeding self-assembly protocol was elaborated and allowed a more uniform distribution of cells throughout the tissue without delineation marks [88].

#### **5.1. Reseeding self-assembly technique**

Ascorbic acid triggers collagen deposition that reaches a plateau level after 2 weeks of fibroblast culture [88]. This time period also correlates with the thickness reached by selfassembled tissue [85]. When fibroblasts reach confluence, the cells begin to secrete and deposit collagen to form the ECM, a step that lasts 2 weeks before collagen synthesis rate decreases. These observations led to the generation of engineered tissues by reseeding of cells instead of sheet stacking (**Figure 1C**). The new reseeding approach is based on the fact that a second layer of fibroblasts seeded onto the first sheet will concomitantly induce a transitory peak of MMP activity and a boost of collagen secretion. The fibroblasts in the first sheet play a role in this

remodeling, and after an additional 2 weeks of culture, the reseeding process results in the generation of a stroma with the same thickness as the one obtained by classical stacking of three sheets produced without reseeding. This dense stroma supported the development and maturation of the epithelium [88]. The reseeding technique offers a remarkable alternative to the classical self-assembly protocol because it is faster and it reduces costs associated with extensive culture medium consumption as well as material [88] (**Figure 3**).

**Figure 3. Graphical illustration of the improvements to the self-assembly technique (SS) when reseeding of cells (RS) is applied**. Reseeding allows the reduction of costs, handling time, and incubator space requirements throughout the steps of cell culture.

#### **5.2. Organ-specific stroma**

Over the years, it has been shown that the origin of mesenchymal cells has a direct impact on the quality of bioengineered tissues. Carrier *et al*. [109] showed that reconstructed human cornea substitutes had great macroscopic and histological differences; especially in the corneal epithelium thickness and differentiation whether dermal fibroblasts or keratocytes (corneal fibroblasts) were used to produce the ECM. Constructs made with dermal fibroblasts were less transparent and lacked ultraviolet absorption characteristics compared to corneal tissues which were produced using autologous keratocytes [109]. The importance of this cross talk was further evaluated by mass spectrometry analyses performed on human stromal and epithelial layers of corneal substitute. Not only is the origin of mesenchymal cell important, but a fully differentiated and stratified epithelium is required for appropriate ECM synthe‐ sis and organization. This reciprocal regulation between the ECM and epithelial components is initiated as epithelial cells adhere to the stroma, they emit a continuous signal that medi‐ ates ECM remodeling accordingly [63].

Modifications to the original protocol have generated near-to-native self-assembled bladder and urethral human tissues. Many improvements include mechanical stimulation [68], the use of autologous human stromal cells, urothelial cells, urine[110, 111], and a new proposed reseeding technique of stromal cells [88]. In particular, the absence of an air/liquid interface and the presence of urine allowed the new bladder mucosa model [110] to be continuously cultured in submerged conditions. Consequently, these modifications generated a bladder model that preserved the best urothelial cell properties and uroplakin distribution [110, 111].
