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BioDesign Research / 2021 / Article

Review Article | Open Access

Volume 2021 |Article ID 9890319 |

Srikumar Krishnamoorthy, Michael F. Schwartz, Lisa Van den Broeck, Aitch Hunt, Timothy J. Horn, Rosangela Sozzani, "Tissue Regeneration with Hydrogel Encapsulation: A Review of Developments in Plants and Animals", BioDesign Research, vol. 2021, Article ID 9890319, 9 pages, 2021.

Tissue Regeneration with Hydrogel Encapsulation: A Review of Developments in Plants and Animals

Received31 Aug 2021
Accepted11 Nov 2021
Published02 Dec 2021


Hydrogel encapsulation has been widely utilized in the study of fundamental cellular mechanisms and has been shown to provide a better representation of the complex in vivo microenvironment in natural biological conditions of mammalian cells. In this review, we provide a background into the adoption of hydrogel encapsulation methods in the study of mammalian cells, highlight some key findings that may aid with the adoption of similar methods for the study of plant cells, including the potential challenges and considerations, and discuss key findings of studies that have utilized these methods in plant sciences.

1. Introduction

Cellular potency is vastly different between plant and animal cells, with plant cells exhibiting a much higher level of regenerative capability. For instance, a large number of plant cell types exhibit pluripotency and totipotency, with stem cell niches (meristems) occurring throughout the plant body [1]. The various plant meristems consist of the apical meristems, of which the shoot and root apical meristem (SAM and RAM) are located at the tip of the shoot and root, respectively, axillary meristem (AM) in the leaf axils, and the procambium, which produces the primary vasculature within plants [2]. Generally, cells derived from almost every plant organ are capable of undergoing differentiation into multiple cell types and thus show significant regenerative capabilities, as opposed to animal cells, which show limited regenerative capabilities varying highly across different organs and species [3, 4]. Moreover, de novo generation of root and shoot meristems has been demonstrated by externally supplying varying amounts of the plant hormones auxin and cytokinin to plant cell culture [3]. This is in contrast to animals, where postembryonic cells are grouped in stem cell niches that occur in a much more localized manner within specific tissues such as the bone marrow, for the sole objective of maintaining and regenerating those specific tissues, and are unable to differentiate into other cell types. These cells are typically multipotent, where they can differentiate into multiple specific cell types that are present in a specific tissue type [5]. Moreover, fully grown animals typically lack pluripotent stem cells [6].

Conventionally, liquid suspension cultures of isolated protoplasts have been the preferred method to understand plant cell regeneration. However, despite the remarkable regenerative capacities of plant cells in nature, plant cells grown in suspension cultures have been demonstrated to have low tissue regeneration efficiency due to cell aggregation, a lack of repeatability, and generally low cell proliferation [7]. In addition, even if animal cells have been shown to have limited regenerative capabilities, researchers have achieved replicable and highly efficient regeneration of animal cells by utilizing techniques such as hydrogel encapsulation [8]. Importantly, hydrogel encapsulation allows for the formation of more physiologically representative microenvironments in tissue regeneration techniques.

Hydrogel encapsulation methods with calcium alginate have been utilized in plant sciences for specific applications such as the cryopreservation of meristems and somatic embryos of Daucus carota, Solanum tuberosum, [9] and Cannabis sativa [10]. However, to our knowledge encapsulation for fundamental studies on cellular regeneration has been limited. In this review, we summarize cell encapsulation methods utilized for the cellular analyses of mammalian cells and as proposed methods for plant cells. Encapsulation of plant protoplasts may allow for better comprehensive understanding of the specific mechanisms behind the cellular potency of plants and pave the way for better mechanisms of replicating plant regenerative capabilities in vitro.

2. Hydrogel-Based Cell Encapsulation, Lessons Learned from Mammalian Cell Studies

For mammalian cells, the primary motivation behind the utilization of encapsulation techniques is due to the established inadequacy of conventional cell culture methods to adequately mimic the complex in vivo microenvironment in natural biological conditions of mammalian cells [8]. For example, monolayer cultures do not adequately represent cell-cell and cell-extracellular matrix (ECM) interactions, which are typically responsible for cellular functions such as cell differentiation, proliferation, gene expression, and protein translation and responsiveness to other stimuli [1113]. In certain cases, cell growth in a two-dimensional (2D) environment has very different growth outcomes than when grown within hydrogel encapsulation, which may represent a more physiologically accurate environment. For example, it has been demonstrated that when human breast epithelial cells are cultured in a standard liquid culture, they express tumor-like growth, while they revert to normal growth when cultured within encapsulated hydrogel environments such as collagen [14, 15].

The utilization of hydrogel encapsulation methods on mammalian cells have resulted in a better understanding of fundamental cellular mechanisms. For instance, the utilization of hydrogel cell encapsulation has demonstrated mechanisms of DNA repair and evasion of apostasies in cancerous cell clusters [16, 17]. The utilization of tissue organoids generated by cell encapsulation has also allowed for new understanding of the interaction between individual cells and the surrounding ECM, thus allowing for a more systematic understanding of cell behavior. For instance, the encapsulation of intestinal stem cells within hydrogels of variable mechanical properties has revealed the presence of mechanosensitive pathways that may regulate cellular survival and subsequent differentiation [18].

3. Hydrogel-Based Cell Encapsulation, Key Differences between Mammalian and Plant Models

The majority of mammal-derived cells are adherent cells, or anchorage dependent [19], and require their adherence to a rigid surface for nominal proliferation and survival as the absence of a culture surface can result in cellular apoptosis due to anoikis [20] and inadequate cell-matrix interactions. This indicates the need for hydrogel materials used in the encapsulation of mammalian cells to consist of the appropriate cell surface receptor-recognition motifs that are essential for cellular anchorage [21]. The majority of plant cells are anchorage independent and therefore do not undergo cellular apoptosis, which offers more flexibility for material selection in the encapsulation of plant cells. Even so, there is evidence that hydrogel matrices that more closely mimic the properties of natural plant tissue environments may yield better cell survival and proliferation [22]. On a similar note, unlike mammalian cells, plant cells comprise a cell wall, whose function is to provide structural support and protection to the cell from biotic and abiotic stresses [23], which can potentially influence the choice of encapsulation material. Protoplasts that lack cell walls due to enzymatic digestion are widely utilized in proteomic and genomic studies and studies of various aspects of plant cell physiology [7]. One key factor of protoplast maintenance to be considered is the normalization of osmotic pressure until the cell walls are fully regenerated, whereas for mammalian cells, the absence of a cell wall requires a more prolonged maintenance of osmotic pressure. Some key differences between animal cells and plant cells that may influence hydrogel encapsulation are illustrated in Figure 1.

There is a need for systematic studies that attempt to quantify effects of various gelation kinetics on the encapsulated plant cells and protoplasts. For instance, alginate is typically crosslinked using divalent cations, especially Ca2+. However, Ca2+ is also known to modulate microtubule organization and thus influence cell wall material regeneration [24], which may influence cellular response. Other factors that are considered in studies involving mammalian cell encapsulation are microstructure of the material and swelling and degradation kinetics [25], which are factors that may need to be considered in plant cell encapsulation as well.

4. Hydrogel-Based Cell Encapsulation, Protoplast Regeneration as Use Case for Plant Cells

A key motivation behind the utilization of hydrogel encapsulation methods in plant sciences are aimed at fundamental studies on processes associated with cell and tissue regeneration, which include the recovery of cell walls, reentry of cell cycles, formation of calli, acquisition of pluripotency, and de novo tissue regeneration [26]. Specifically, cells do not have high proliferation in liquid suspension cultures and are also not an adequate platform for studying the molecular mechanisms that influence the regeneration of cells [27]. Hydrogel encapsulation within thin alginate layers and thin alginate films (μm) have been utilized for the immobilization of Daucus carota protoplasts. This allowed for the study of the effects of various culture conditions on the embryogenesis of these protoplasts [28].

Sinha and Caligari have shown the enhanced rate of protoplast division of recalcitrant lupin protoplasts when encapsulated within hydrogel droplets when compared to suspension cultures [22]. Specifically, protoplasts were isolated from cotyledons of two-week-old Lupinus albus seedlings and cultured in four different culture modes, as illustrated in Figure 2, to compare development in liquid suspension with hydrogel encapsulation: (i) suspension over a rigid substrate, (ii) suspension over an agarose layer, (iii) protoplasts embedded within a single layer of agarose hydrogel, and (iv) protoplasts encapsulated within agarose droplets. Subsequently, the division and the morphology of the protoplasts in the different aforementioned cases were quantified. Moreover, protoplasts encapsulated in additional hydrogels with different gelation kinetics were also compared, including different concentrations of gellan gum and sodium alginate. It was found that agarose at low concentrations were more conducive to protoplast division than other hydrogels, and it was postulated that there might be a direct relationship between hydrogel firmness and area of contact between the protoplasts and surrounding matrix. Generally, it was found that encapsulation within agarose droplets resulted in higher rates of mitosis, while alginate embedding inhibited protoplast division. It was suggested that the utilization of a continuous matrix as provided by hydrogel droplets mimics a meristematic tissue and the natural immobility of plant cells within the tissue.

Similar results were noted by Deryckere et al. when they compared three different modalities of encapsulation for plantlet regeneration of Cichorium protoplasts (i.e., solid media, liquid suspension, and encapsulated beads) and demonstrated enhanced regeneration in low melting point agarose beads compared to the other two modalities [29]. Specifically, the protoplasts from leaves of 11 Cichorium genotypes were isolated and cultured independently in the three aforementioned encapsulation methods, after which the cell viability and cell division was tracked. The cells in the liquid suspension clustered together, which in turn reduced cell viability due to the transfer of toxic compounds from one cell to another within the clusters. This was also confirmed by the detection of the build-up of anthocyanins in the liquid suspension. The fully solid agar media was impacted by the inability to be regularly refreshed, and this caused a lowering of osmotic potential due to dehydration, which lowered viability and prevented any cell division from happening. On the other hand, the cells that were encapsulated in the agar beads thrived optimally and showed the highest rates of division, due to being able to be refreshed with fresh media regularly, and the homogenous distribution of cells in the agar beads prevented cluster formation from happening. This shows that hydrogel encapsulation requires a multifaceted consideration of factors such as mode of encapsulation and may also potentially be a method to control cell-to-cell interactions, such as cluster formation.

Another aspect of hydrogel encapsulation that might influence the growth of encapsulated protoplasts is the choice of hydrogel material used for encapsulation, and the genotype from which the protoplasts are isolated. For instance, Prange et al. established a regeneration protocol from protoplasts of three wild species of Cyclamen, Cyclamen graecum, Cyclamen mirabile, and Cyclamen alpinum and compared the cell growth responses in two different embedding agents, agarose and alginate, and observed different cell responses from protoplasts of different genotypes [30]. The key results from this study are illustrated in Figure 3. Cyclamen alpinum cells showed normal development in alginate but were elongated and bulged in agarose, with irregular cell wall strengths. However, microcalli formation was only observed after 6-10 weeks of culture in both embedding media. Cyclamen graecum protoplasts exhibited similar circular morphology in both alginate and agarose and showed similar rates of division and high formation of microcalli within a few days. In contrast, Cyclamen mirabile protoplasts showed significantly higher division rates in agarose when compared with alginate and showed irregular cell wall regeneration in agarose. This highlights the need to use a system-level approach to selecting a hydrogel material for the encapsulation of protoplasts—a specific hydrogel may elicit a different outcome in different genotypes of even the same species of plant, and it is necessary to make careful consideration of the desired outcome prior to the selection and utilization of a hydrogel material. Moreover, hydrogel selection is not a trivial choice and requires further cell-level studies to understand the effect of different hydrogels on protoplast behavior of different species and genotypes.

The utilization of hydrogel encapsulation methods can allow for a better in-depth understanding of protoplast behavior during processes such as division and differentiation, as shown in Figure 4. Sakamoto et al. have demonstrated the utilization of sodium alginate for encapsulating Arabidopsis thaliana leaf protoplasts, followed by culturing in protoplast callus growth medium (PCIM) that was supplemented with 2,4-D and thidiazuron as auxin and cytokinin, respectively [31]. Alginate encapsulation allowed for detailed analysis of the protoplast differentiation progression for better understanding of cell reprogramming and shoot regeneration. For instance, time-lapse imaging of the encapsulated protoplasts allowed for closer examination of early morphological changes and revealed that the protoplasts undergo their initial cell division between 4 and 7 days of encapsulation. Moreover, it was observed that the protoplasts undergo variable changes such as anisotropic elongation or shrinkage [31]. Changes in vacuolar morphology was tracked by time-lapse imaging of protoplasts carrying monomeric GFP- (mGFP-) tagged VACUOLAR H+-PYROPHOSPHATASE 1 (VHP1) (pVHP1:VHP1-mGFP), and it was observed that fresh protoplasts consisted of one primary vacuole within the cell volume, followed by the appearance of strand-like structures before and during cellular elongation, followed by excessive compartmentalization of the vacuoles through successive divisions of the cells. It was discovered that the initial cell division was achieved by the biosynthesis of auxin by the activation of G2/M phase genes mediated by MYB DOMAIN PROTEIN 3-RELATED (MYB3Rs).

The study of transcriptome changes in protoplasts may aid with the better selection of material for encapsulation. As shown in Xu et al., it demonstrated that increases in chromatin accessibility results in the stochastic activation of gene expression and enhances regeneration of protoplasts, which was visualized by the live-imaging of early protoplasts encapsulated in alginate [32]. Arabidopsis thaliana mesophyll protoplasts were isolated from the following transgenic lines: pPLT7::PLT7-YFP, pWOX5::GFP, pBBM::BBM-YFP, pWUS::3xVenus-N7, pDRN::mCherry-N7, pDRNL::3xVenus-N7, pCLV3::mCherry-N7, pWOX2::NLS-DsRed3, pUBQ10::WUS-GR, and p35S∷DRN-GR, and from wild-type plants, which was followed by changes to the transcriptome during regeneration of individual protoplasts. This was supplemented by the live imaging of early protoplasts by the embedding of these protoplasts on a polypropylene grid and subsequent encapsulation in alginate, followed by culturing in protoplast induction media (PIM). Long-term live imaging revealed that 0.5% of the protoplasts exhibited regeneration capabilities after 56 days of culture and formed microcalli that were greater than 200 μm. To identify the specific genes that promoted totipotency acquisition, transcriptome profiles were obtained at different stages of the process, namely, immediately after protoplast isolation, and after 4, 11, 22, and 30 days of culture in PIM, and were compared with the transcriptome profiles of intact leaf cells. It was demonstrated that protoplast isolation by the digestion of cell walls tends to be a significant cause of ectopic activation of gene expression and is illustrated in Figure 5. Isolation of protoplasts tends to increase the variation in gene expression among cells, and the stochastic expression tends to endow cells with heterogeneous fates, including endowing a small portion of the cells with pluripotency or totipotency. Interestingly, the genes that were activated stochastically were compared with established gene expression profiles from different plant regeneration methods. For instance, WUSCHEL (WUS) was observed to be necessary for protoplast regeneration, with an increase in overexpression of WUS being correlated with increased regeneration efficiency. PLETHORA (PLT) genes have been previously established to be activated during regeneration due to mechanical injury to plant organs [33]. However, no PLT activation was observed during protoplast isolation, which indicated that wall digestion was the primary cause of gene expression, rather than mechanical damage to the leaves that are required prior to protoplast isolation. Additionally, prior studies have established WUS expression as being essential to shoot regeneration by establishing a stem cell niche [34].

5. Potential Challenges with Hydrogel Encapsulation Methods

While the aforementioned studies show the potential of utilizing hydrogel encapsulation as a tool for plant cell culture, there are some critical disadvantages associated with the technique that need to be considered. For instance, hydrogel encapsulation may potentially hinder downstream processes such as transcriptomic profiling due to the need to release encapsulated cells. This may require additional enzymatic degradation of the bulk matrix, such as agarases on agarose-derived hydrogels [35], or collagenases on collagen-derived hydrogels [36]. The release of encapsulated cells may also be facilitated by utilizing reversible crosslinking methods, which may allow for relatively simple liquefaction of crosslinked hydrogels. For instance, alginates are crosslinked using ionic crosslinking strategies, which are reversible by using calcium chelators such as sodium citrate [37]. Similarly, thermally crosslinked gelatin, and other related hydrogels, can be liquefied by simply varying the external temperature to the liquefaction temperature [38]. However, changes in temperature outside of an optimal window is known to cause changes in gene expression in protoplasts [39], and this must be considered before utilizing temperature-based liquefaction methods.

One of the key motivations behind using hydrogel encapsulation is being able to achieve high rates of cell division in encapsulated protoplasts and cells. However, the presence of a solid matrix may also hinder the rate of division and proliferation of some protoplasts of some species. For instance, Chrysanthemum indicum protoplasts have been documented to have significantly higher rates of proliferation in liquid culture when compared to hydrogel encapsulation. This may also be potentially due to accumulation of cellular waste within the hydrogel matrices [40]. Similarly, regeneration of plants from Populus×beijingensis was also found to be significantly higher in liquid culture, due to the ability to easily replenish fresh media. This allows for the reduction of medium osmolarity while also supplying new nutrients for the cells, which in turn can enhance division frequency [41]. Similar results were observed in the cultivation of Kalanchoe blossfeldiana protoplasts for plant regeneration, with the liquid culture offering a more direct approach to replenishing nutrients [42], and in the culture of leaf- and calli-derived protoplasts of Albizia julibrissin which showed improved division and regeneration in liquid suspension as opposed to hydrogel encapsulation in agar [43]. Thus, cases such as these may require the utilization of additional media supplantation around hydrogel constructs in order to dilute such molecules.

6. Conclusions

Hydrogel encapsulation is a widely utilized method in the study of cell behavior of mammalian cells that has been gaining traction in studies on plant development. More systematic studies on hydrogel encapsulation may provide new avenues for fundamental research on plant cell behavior and thus on plant development. Specifically, studies such as cell-to-cell signaling, regeneration, and organ development may be made more possible by the utilization of hydrogel encapsulation techniques on plant cells and protoplasts and may pave the way for specific applications such as improved crop development and hybridization. While there are challenges associated with encapsulation, more extensive research is required to specifically highlight the relationship between hydrogel matrix properties, and cellular behavior of encapsulated plant cells, and thus enable more fundamental plant development research by offering up pathways for the use of novel scaffolds and matrices on different species and genotypes of plants. Moreover, the utilization of hydrogel encapsulation may also be a key enabling technology to easily observe gene expression due to different cellular processes, by providing a platform for isolating and observing specific cells and tissues.

Conflicts of Interest

The authors declare that they have no competing interests regarding the publication of this article.


This work was supported by the NSF EAGER grant (MCB #2039285) to TJH and RS.


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