Directed assembly of microgels containing cells to create three-dimensional tissue structures
Based on the article by Yanan Du et al. "Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs"
(PNAS July 15, 2008 vol. 105 no. 28 9522-9527).
Scientists from Harvard and Cambridge Universities and the Massachusetts Institute of Technology have developed a method of bottom-up controlled assembly of hydrogel microparticles loaded with cells to create tissue structures. The proposed process is based on the ability of multiphase liquid systems to minimize the surface area of the phases and, accordingly, the free surface energy of the phase separation. The complexity of the microarchitecture of such hydrogels is regulated by changes in external influences.
Most living tissues consist of repeating fragments, which are complexes of cells of various types that have a strictly defined three-dimensional microarchitecture and perform functions specific to this tissue. When creating artificial fabric structures, the reproduction of structural features of natural tissue plays an important role in ensuring the performance of tissue-specific functions. However, most approaches of tissue engineering are based on self-assembly of complex cellular structures on biodegradable frameworks (according to the "top-down" principle. Imitation of the structure of natural tissues with this approach does not always occur correctly enough. The use of hydrogels provides new opportunities to solve this problem by creating biocompatible hydrogel structures ranging in size from <1 micron (intracellular level) to >1 cm (tissue level).
The attractiveness of microgels for tissue engineering is due to their physical properties (strictly defined particle shape, mechanical strength, biodegradability, etc.) and biological parameters (biocompatibility, similarity to the natural extracellular matrix and the ability to retain cells in natural tissue concentrations). Currently, the use of microgels in tissue engineering is conducted according to two principles: "descending" and "ascending". The descending principle allows you to control the microstructure parameters (such as size and shape) of relatively large fragments of hydrogels, and the ascending one allows you to create larger tissue structures by combining small blocks (usually loaded with microgel particles cells).
To date, the upward assembly of microgels is carried out using photolithography, which allows the microgel layers to be positioned in a strictly defined way, as well as by arbitrary fusion of microgel particles containing cells or physical manipulations of individual microgel particles. The limitations of the arbitrary self-assembly method include the inability to control the location of hydrogel particles and the structure of the final product, and the other two approaches are multi-stage, time-consuming and unsuitable for large-scale production. These difficulties necessitate the search for an ascending method that provides a directed self-assembly of the required amount of material.
For the first time, a method of self-assembly of particles whose size did not exceed several millimeters into clearly structured two- and three-dimensional objects by minimizing the free energy at the interface of liquid phases was proposed by Whitesides et al. However, their approach is not applicable to tissue structures due to the cytotoxicity of the materials used and the rigidity of the physical conditions necessary for such a process.
The authors of this article propose a fundamentally new approach to the self–assembly of microgel particles containing cells into tissue structures by using the "hydrophobic effect" - the thermodynamic tendency of multiphase liquid systems to minimize the interface of phases, due to which the free energy of the interface of hydrophobic and aqueous phases, proportional to the area of the interface, tends to a minimum.
To test the hydrophobic effect, the authors synthesized particles of polyethylene glycol (PEG) microgel using photolithography, which were placed in a hydrophobic phase from mineral oil. After that, larger microgel structures were assembled using controlled mixing (Fig. 1).
As a result, the researchers obtained four types of structures: linear, branched, disordered and with fragment displacement. Since the hydrophobic effect minimizes the interface between the water and oil phases (water is divided into spherical droplets), the optimal geometric solution in this case is the formation of cubic structures consisting of densely packed rectangular fragments of hydrogel. However, the most interesting from a practical point of view was the fraction of aggregates forming linear segments, which, due to the small length of the chains, provide the most acceptable result from a thermodynamic point of view.
Fig. 1. Schematic diagram of the assembly process of microgel complexes. The microgel particles synthesized by photolithography were introduced into a container with mineral oil and manually mixed with the tip of a pipette. As a result, four types of structures were obtained: linear, branched, disordered and with fragments offset. Secondary cross-linking of fragments was carried out by exposure to ultraviolet radiation. (The price of dividing the scale ruler is 200 microns).
The effect of the mixing speed and duration, as well as the presence of a surfactant, was assessed by the characteristics of rectangular microgel particles (Fig. 2). Faster mixing provided a larger fraction of linear complexes (up to 30% in 15 seconds) (Fig. 2A). An increase in the duration of the mixing time caused an asymptotic growth of the fraction of linear complexes during the first 60 seconds, after which the growth of the fraction stopped (Fig. 2B).
Fig. 2. Optimization of microgel assembly. The effect of (A) the mixing speed (fast, medium and slow), (B) the duration of mixing and (C) the addition of surfactant.
Surface tension is the driving force behind the aggregation of microgel particles by minimizing the surface of their contact with the oil phase. To study the effect of changing this parameter on the aggregation process, the authors changed the surface tension of the phase interface by adding an emulsifier (Twin 20). As expected, the addition of a surfactant significantly reduced the driving force of directed aggregation of microgel particles (Fig. 2C), which was expressed in a decrease in the fraction of linear and branched complexes and an increase in the fraction of disordered aggregates as the concentration of the emulsifier increased.
The researchers also analyzed the effect of changes in the size of individual microgel particles on the formation of complexes. At a constant height of the fragments (150 microns), their length and width were increased from 200 to 1000 microns in increments of 200 microns. At the same time, smaller particles ensured the production of large fractions of linear and branched complexes and smaller fractions of disordered structures than large microgel particles (Fig. 3A). Perhaps this is due to the higher hydrodynamic force and drag force acting on larger microgel particles and outweighing the forces associated with the hydrophobic effect. Moreover, microgel particles with higher size ratios form chains with a large number of links (Fig. 3B), which minimizes the surface area of the phase interface.
Fig. 3. The effect of microgel particle sizes on their aggregation. (A) The composition of complexes and (C) the average length of chains of linear, branched and disordered aggregates.
Despite the fact that the two-phase assembly process allows controlling the formation of microgel complexes, the resulting structures are unstable outside the oil phase. To stabilize the bonds between the microgel particles, a secondary "binding" stage was performed – the material was exposed to ultraviolet radiation for 4 seconds.
To assess the possibility of using the developed process for the needs of tissue engineering, fibroblasts of the NIH 3T3 line were encapsulated in microgel particles. Immediately after encapsulation, most of the cells remained viable. Moreover, the use of the developed assembly process of larger structures preserved the viability of cells at a sufficiently high level (Fig.4A). To the surprise of the authors, a small number of cells died at the mixing stage of the two-phase system, which may have been caused by water-soluble pollutants contained in the hydrophobic phase. As expected, the duration of exposure to ultraviolet radiation had a significant impact on cell viability, which indicates the need to minimize both stages.
Fig. 4. Microgel particles loaded with cells. (A) Phase-contrast and fluorescent images of cell-containing (NIH 3T3) microgel complexes after the stages of primary and secondary binding, respectively. (C) Cell viability after each of the stages of formation of microgel aggregates.
Fig. 5. Controlled assembly of microgel fragments, carried out according to the "key-lock" principle. (A) Fluorescent images of cross-shaped microgel particles stained with FITC-dextran. (B) Elongated microgel particles stained with Nile red dye. (C-H) Phase-contrast and fluorescent images of key-lock complexes with different quantitative ratios of cruciform and elongated particles. (I, J) Fluorescent images of a microgel complex consisting of cruciform particles containing red-labeled cells and elongated particles containing green-labeled cells. (The price of dividing the scale ruler is 200 microns).
When using previously existing methods, despite the possibility of controlling the size and architecture of the final microgel complexes, the connection of individual particles occurred randomly. For example, it was impossible to control the adjacent arrangement of particles of different types of microgel. To demonstrate the suitability of the new approach for creating more complex structures with specified properties when creating microgel particles, the authors used the so-called "key-lock" approach, which ensures the mutual arrangement of two types of microgel particles in the final complex. As can be seen in Fig. 5, microgel particles of a cruciform and elongated shape (Fig. 5 A, B) can be directionally combined in such a way that one cruciform particle connects with one, two and three elongated particles (Fig. 5 C-H). The proposed method allows obtaining up to 10% of key-lock complexes without any additional optimization. In addition, the authors demonstrated the suitability of this approach for creating combined cell cultures. To do this, they encapsulated cells labeled with red and green labels into cross-shaped and elongated microgel particles and created miniature tissue structures from two types of cells (Fig. 5 I, J).
Thus, the authors have developed a method that uses the thermodynamic properties of multiphase liquid systems to assemble hydrogel microparticles loaded with cells.
They demonstrated the possibility of controlling the assembly of hydrogel particles using forces involved in minimizing the free surface energy of the phase separation. This bottom-up method of directed assembly of microgel particles containing cells is an effective approach to the creation of three-dimensional tissue structures, the performance of which is quite easy to adjust. Taking into account the growing possibilities of photolithographic approaches, the authors believe that the new method will make it possible to create highly structured complexes, the production of which using traditional microengineering systems is too difficult and time-consuming.
Evgeniya Ryabtseva
Portal "Eternal youth" http://www.vechnayamolodost.ru06.10.2008