Page 77 - IJB-5-1

3D tissue hybrid biofabrication
molecules, mainly the cadherins and connexins [42] . The different, this method has common goals with regular
cell cytoskeleton [43] and metabolism may also have bioprinting, such as the use of a nozzle to place the cells
indirect roles but probably are less important than direct in a layer-by-layer formed 3D pattern, as well as the
cell-cell attachment. A good argument that spheroid fusion computer-assisted design and operation.
is relatively well understood is the ability to simulate it Recently, several pre-clinical examples of “Kenzan”
by computer modeling on several platforms, for example, scaffold-free biofabrication have been reported:
CompuCell3D , initially developed around this type of Surgically-robust small diameter vascular grafts [61] ,
[44]
applications [45] . We recently expanded a similar model tracheal [62] and urethral [63] tubes, neural bridges ,
[64]
to capture the role of oxygen during spheroid fusion in beating cardiac patches [65] , liver buds [66] , and gastric
larger structures . diaphragm . However, the Kenzan method is not without
[63]
[46]
Spheroid generation is best achieved by placing the cells its own limitations, as we commented previously .
[7]
on non-binding surfaces either in large flat or small, round Among the more significant ones are: (i) Inability to
wells [47] . Cells sedimenting on a surface that offers no import anatomically-correct 3D images; (ii) dependence
anchorage will adhere to other cells thus forming spherical of this method on the cells’ propensity to make spheroids
clusters, a tendency that can be promoted by small inverse of required size (commensurate with the inter-needle
pyramidal depressions [48] . Depending on geometry and distance); (iii) secretion of a matrix strong enough to keep
coating, the latter allows for homodisperse spheroid sizes, the construct compact; and (iv) length of the constructs
while larger non-binding wells might give rise to numerous limited to that of the microneedles.
differently sized spheroids. The spheroids so formed then
have to be repositioned to induce them to fuse into 3D 3. Hybrid Biofabrication
structures. To this end, the spheroids can be placed in Since the inception of bioprinting, those involved in
molds where they form 3D structures by fusing . Tissue its development rightly appreciated the difficulties
[41]
models of cartilage [49] , or cardiac patches prepared from derived from the use of a biomaterial and contemplated
spheroids fused by flotation on culture medium , as well alternatives (for instance, ). These consist of using cell
[50]
[64]
as vascular rings and tumor models [52] assembled by spheroids as building blocks for direct assembling the 3D
[51]
magnetic force, have been described. As a variant, thicker construct, even if the spheroids themselves may require
honeycomb-shaped cell toroids prepared in molds, then temporary support of some sort (such as “fugitive”
stacked in register, and fused in larger constructs were hydrogels, or mechanical assistance).
proposed for improved distribution of nutrients, even in Thus, some of the properties of scaffold-free
the absence of a bona fide vascularization . biofabrication could be complemented, at least in part, by
[53]
2.2.2 Pick and Place including biomaterials into the “scaffold-free” constructs.
For example, these biomaterials could compensate
Alternatively, spheroids can be individually manipulated for the slower intrinsic secretion of an ECM by some
by dedicated instruments (such as the “Fabion” cell types, when prepared as spheroids. Alternatively,
[54]
bioprinter ), and placed in a pattern on support where “classical” bioprinting may also benefit from several
they fuse and form hollow or mixed massive structures. principles of the scaffold-free approach. Combined,
Interestingly, the targeted placement of spheroids on this technological inter-breeding establishes the field of
support (which some authors address as “biopaper” [55-57] ) “hybrid” biofabrication.
refers back to the printing analogy. The use of sacrificial hydrogels for holding spheroids in
To facilitate the formation of 3D constructs from place, until fusion and during a “post-printing maturation”
spheroids, a method was needed to keep them in contact phase [54,65] , is a typical example of hybrid bioprinting.
long enough to effect fusion, and at the same time to allow Combination of spheroids with hydrogels could be
the cells to produce their own ECM [58] . The companies profitable for regular bioprinting as well if instead of
Organovo [59] and 3D Bioprinting Solutions are single-cell suspensions pre-formed spheroids are mixed
[54]
performing this step on their bioprinters using “fugitive” within the bioink. In practice, this solution has been
hydrogels as supports, which are removed after the used to increase the human adipose-derived stromal cells
spheroid fusion process. survival and promoted their ability to differentiate after
bioprinting . The bioprinter can be also used to prepare
[66]
2.2.3 Pick and Skewer
cells mixed in bioinks of alginate [67] or collagen [68] in
An ingenious solution to spheroids assembling problem droplet form, as an efficient method of cell encapsulation,
has been developed in Japan [60] . Essentially, this is for subsequent in vitro or in vivo deployment. Moreover,
based on using a spheroids-assembling robot, which the addition of fibrillary materials to hydrogels has been
skewers them on a rectangular array of stainless steel shown to improve their mechanical properties, thus
micro-needles (“Kenzan)” . Although technologically generating stronger tissue-like constructs [30,69,70] .
[7]
4 International Journal of Bioprinting (2019)–Volume 5, Issue 1