This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.
To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.
Traditional cell culture on flattened two-dimensional (2D) surfaces, such as a culture dish or multi-well plates, can hardly elicit cell behaviors close to their native states. Accurate recapitulation of native cellular microenvironments, which comprise of various cell types, extracellular matrices and bioactive soluble factors in three-dimensional (3D) architectures1,2,3,4, is essential to construct biomimicking tissues in vitro for applications in tissue engineering, regenerative medicine, fundamental biology research and drug discovery5,6,7,8,9.
In lieu of 2D cell culture, 3D cell culture is widely used to advance biomimetic micro-architectural and functional features of cells cultured in vitro. A popular 3D cell culture method is to aggregate cells into spheroids7,8,9,10. Cellular spheroids could be injected to injured tissues with enhanced cellular retention and survival in comparison to injection of dispersed cells. However, non-uniform spheroid sizes and inevitable mechanical injury imposed on cells by fluid shear force during injection lead to poor cell therapeutic effects11,12,13. Similarly, the inherent non-uniformity during aggregation of spheroids has made their translation to 3D cell-based high-throughput drug screening challenging10.
Another method for 3D cell culture is achieved with the assistance of biomaterials, which typically encapsulates cells in aqueous hydrogels or porous scaffolds. It allows for greater flexibilities in constructing 3D architectures. For therapy, cells encapsulated in bulk scaffolds are usually delivered to animal body via surgical implantation, which is invasive and traumatic, hence restricting its wide translation to bedside. On the other hand, aqueous hydrogels enable minimally invasive therapy by injecting cells suspended in hydrogel precursor solution into animal bodies, allowing in situ gelation via thermo-, chemical or enzymatic crosslinking11. However, as cells are delivered whilst the hydrogel precursors are still in an aqueous state, they are also exposed to mechanical shear during injection. Not only so, chemical or enzymatic crosslinking during in situ gelation of hydrogel could also impose damage to cells within. For drug screening, biomaterial-assisted cell cultures face problems with uniformity, controllability and throughput. Using hydrogels, cells are typically involved during gelation, by which the process may affect cell viability and function. Gelation during cell seeding also hampers usage by most high-throughput equipment, since the hydrogel may need to be kept on ice to prevent gelation before cell seeding, and the hydrogel might jam dispensing tips, which are usually very thin to ensure accuracy for high-throughput screening. Pre-formed scaffolds could potentially separate biomaterial fabrication procedures from cell culture, however most scaffold-based products are available as bulk materials with relatively lower throughput14.
To overcome some of the shortcomings of current 3D culture methods, we have developed a microfabrication-cryogelation integrated technology to fabricate an off-the-shelf and user-friendly microcryogel array chip15. In this protocol, gelatin is selected to exemplify the microcryogel fabrication technique as it is biocompatible, degradable, cost-effective, and no further modification is required for cell attachment. Other polymers of natural or synthetic sources could also be used for fabrication, depending on the application. Via this technology, we can fabricate miniaturized and highly elastic microcryogels with controllable size, shape and layout. When loaded with a variety of cell types, 3D microtissues could be formed for various applications. These unique features enable desired injectability, cell protection and site-directed retention after injection in vivo for enhanced therapeutic effects. Not only so, the microcryogels could be further processed to form 3D microtissue arrays that are compatible with common laboratory equipment and instruments to realize high-throughput cell culture for versatile drug screening and other cellular assays. Herein, we shall detail the fabrication process of microcryogels and its post-treatment as individual 3D microtissues or 3D microtissue arrays for two important applications, cell therapy and drug screening, respectively10,15.
Animal experiments followed strict protocol approved by the Animal Ethics Committee on the Center of Biomedical Analysis, Tsinghua University. Under approval of Ethics Committee, human adipose tissue was obtained from Department of Plastic Surgery of Peking Union Hospital with informed consent from the patients.
1. Fabrication of 3D Microcryogels
2. Harvesting Individual Microcryogels to Form Injectable 3D Microtissues for Treatment of CLI
3. Assembly of Microtissue Array Chip for High-throughput Drug Screening
Fabrication and characterization of microcryogels for 3D microtissue formation.
According to this protocol, microcryogels were fabricated to form the 3D microtissues and individual microcryogels or microcryogel arrays, and were applied to regenerative therapy and drug screening, respectively (Figure 1). Microstencil array chips fabricated from PMMA were applied as micromolds for microcryogel array chips. Variable geometrical designs could be prepared for the microstencil array chip. We chose a representative 45 mm x 14 mm microstencil array chip as an example, which contained various shapes (i.e., circle, ellipse, triangle, and clover) and a circle-shaped microstencil array chip with different sizes (diameters = 100, 200, 400 and 800 µm). To enhance the visibility of micromolds on the array chips, light epi-illumination images were observed (Figure 2A, B). Microcryogels harvested from the array chips exhibited desired shapes and sizes (Figure 2C, D). Such microcryogels with desired geometrical features could possibly be applied as templates to form different cellular units that mimic certain architectures of native tissues. The harvested GMs (gelatin microcryogels) had pre-defined shapes and sizes (Figure 3A). SEM observation demonstrated that microcryogels contained interconnected macroporous structures with pore sizes in the range of 30 – 80 µm (Figure 3B).
Enhanced injectability of hMSCs-loaded microtissues for improved ischemic limb salvage
Using the programmable syringe pump integrated with a digital force gauge14, the injectability of GMs was quantitatively assessed. At a flow rate of 1 mL/min, the GMs with a density of 1,000 microcryogels per mL were injected under 6 N, which was lower than the clinically acceptable force of 10 N20 (Figure 3F). Basing on cell protection enabled by GMs, hMSCs in GMs had high viability and maintained great proliferative capacity after injection during 5 days of culture (Figure 3H).
The mouse limb ischemia model was chosen to assess the therapeutic efficacy of the injectable hMSC-loaded microtissues. Physiological status of ischemic limbs was examined 28 days after surgery (Figure 4A). No limb salvage was observed in the sham group or GMs control group. In the 105 free cell treatment group, 50% total toe amputation, 25% partial toe amputation, and 25% partial limb amputation were observed within 7 days, resulting in 80% limb loss and 20% total toe amputation after 28 days. In contrast, microtissues treatment with 105 hMSCs achieved improved limb salvage (75%) with only 25% mice showed spontaneous toe amputation after 28 days. 106 hMSCs, the minimum effective cell number used in most previous studies, was chosen as the positive control21. Only 2 of the 4 mice showed limb salvage, but all had minor necrosis.
Blood perfusion was monitored and assessed in the aid of indocyanine green (ICG), an FDA approved angiographic contrast agent. The result showed that fluorescence signals appeared in the microtissue-treated mice and in the 106 free hMSCs-treated mice. There is no evident fluorescence signal in the ischemic hindlimbs in the sham or GM group until day 28 (Figure 4B).
These results further confirmed that 3D microtissue-assisted hMSCs therapy achieved superior therapeutic effects for CLI treatment which represents the minimum effective dosage for cell-based therapy in the mouse model so far.
High-throughput drug cytotoxicity screening on 3D microtissue array chip
A ready-to-use 3D microcryogel array for on-chip cell culture could be easily fabricated by retaining microcryogels on the PMMA chip after lyophilization and combining with the corresponding well-array chip with biocompatible adhesive tapes (Figure 1 and Figure 5A). In this two-part cell culture array, the top well-array chip served as reservoirs for the culture medium, drug solutions and assay reagents, while the cells were cultured in the 3D microcryogel immobilized on the bottom array chip. The adhesive tapes between the top and bottom array chip allowed generation of 384 individual wells for high-throughput 3D cell culture (Figure 5B), hence providing a practical tool for drug discovery.
As described in the protocol, 3D microtissue array was formed by directly seeding cells into microcryogels before adding medium to the reservoirs. Using RFP-labeled NIH-3T3 cells, we demonstrated that the cells were uniformly distributed with multi-layers within the 3D architecture of microcryogels (Figure 5C, D). SEM images showed that cells adhered firmly to the walls of the pores and even exhibited extended filopodia along or across adjacent walls of macropores in the microcryogels (Figure 5E).
We then showed the feasibility of applying this 3D microtissue array for high-throughput drug testing using two cancer cell lines and two compounds. Hepatocellular carcinoma cells (HepG2) were treated with Doxorubicin while non-small-cell lung cancer cells (NCI-H460) were treated with IMMLG-8439, a new tumor inhibitor. Five to nine discrete concentrations of each drug were administered to six adjacent wells as replicates, with 0.1% DMSO in culture medium as the negative control. A cytotoxicity assay was similarly performed for cells cultured in traditional 2D multi-well plates. After 24 h of incubation, the cell viability assay was used to assess the drug responses of cells in both 2D and 3D. Drug response curves were plotted using normalized cell viability rates at different drug concentrations, and the IC50 was then interpolated from these curves. A higher IC50 value would indicate that cells are more drug-resistant. From Figure 5F and 5I, we observed a significant increase in drug resistance when cells were cultured on 3D microtissue array than in 2D. The IC50 of Doxorubicin against HepG2 cells reached 165.959 µM, relative to 18.239 nM on 2D; the IC50 of IMMLG-8439 on NCI-H460 cells was similarly elevated to 331.894 nM in 3D while only requiring 1.294 nM on 2D. Such observation was in concordance with reports of increased drug resistance in 3D culture over 2D culture by other researchers22,23.
We attributed such increase in drug resistance to the complexity of the 3D microenvironment compared to the planar configuration of 2D culture. SEM images revealed that HepG2 cells gathered as spheroids decorating the surfaces of macropores in the microcryogel. These cells are tightly clustered and such enhanced cell-cell interaction could be a source of drug resistance in HepG222. It was also interesting to note that these cell clusters were not freely suspended spheroids as they still maintained some adhesion to the matrix (Figure 5G, H). Conversely, epithelial-mesenchymal-transition (EMT) was speculated to have occurred when the non-small lung cancer cells, NCI-H460, were cultured on 3D microcryogels. NCJ-H460 cells spread out like fibroblasts (Figure 5J, K) instead of clustering like HepG2. Hence, we speculated that the increase in drug resistance could be a result from a transition of epithelial NCI-H460 cells to a more malignant state18,19,20,21,22,23.
Figure 1: Schematic of 3D Microtissue Fabrication and Application in Regenerative Therapy and Drug Screening. Briefly, size and shape-controllable microcryogel chip was fabricated on an array PMMA chip by cryogelation of gelatin. The microcryogel chips can be harvested off-chip as individual microcryogels and further, individual microcryogels can be auto-loaded with cells and cultured to form 3D microtissues for injectable regenerative therapy. Another application of microcryogel chips is to assemble with a reservoir array chip and then further, load cells and culture into 3D microtissue arrays for high-throughput drug screening. Please click here to view a larger version of this figure.
Figure 2:Microstencil Array Chips. (A, B) Photographs of two PMMA microstencil chips containing arrayed microwells with different shapes (i.e., circle, ellipse, triangle and clover) and circular shape with different sizes (diameter: 100, 200, 400 and 800 µm), respectively (A), and two corresponding microcryogel array chips (B). (C, D) Microscopic images of the individual microcryogels harvested from the two microcryogels array chips. Scale bar = 500 µm. This figure has been modified with permission from reference14. Please click here to view a larger version of this figure.
Figure 3: Characterization of 3D Injectable Microtissues. (A) Photographs of harvested microcryogels. (B) Scanning electron micrograph (SEM) images of microcryogels showing interconnected and macroporous structures. (C, D) Fluorescence microscopic and 3D reconstructed confocal images of hMSCs-loaded microtissues stained by live/dead and rhodamine phalloidin. (E) Quantification of hMSCs autoloading and proliferation in GMs with different initial loading densities. (F) Real-time injection force measurement curves for triple injections of 1,000 GMs in 1 mL of 15% (wt/vol) gelatin solution at 1 mL/min injection rate (1,000-1-15%). (G) Live/dead cell viability assay of hMSCs-loaded microtissues pre-injection and post-injection. (H) Proliferation of hMSCs loaded in GMs post-injection after 1, 3, and 5 days of culture (n = 3). *p< 0.05, one-way ANOVA compared to day 1. Data are presented as mean ± SEM. This figure has been modified with permission from reference11. Please click here to view a larger version of this figure.
Figure 4:Improved Salvage and Enhanced Angiogenesis in Ischemic Hindlimbs Treated with 3D Injectable Microtissues. (A) Representative photographs of sham (n = 4), blank microcryogels (n = 4), free hMSCs (105) (n = 8), hMSCs (105)-loaded microtissues (n = 8), and free hMSCs (106) (n = 4) at 0, 3, 7, and 28 day after treatment. (B) Fluorescence images obtained 100 s after ICG injection on day 28. This figure has been modified with permission from reference11. Please click here to view a larger version of this figure.
Figure 5: 3D Microtissue Array for High-throughput Drug Screening. (A) Photograph of 3D microtissue array in 384-multi-well format after assembly, and (B) resazurin cell viability assay performed on the array. (C) RFP-3T3 cells within microcryogel post-seeding (scale bar = 200 µm). (D) 3D reconstruction of nuclei staining depicting homogeneous distribution of cells in multi-layers in microcryogel. (E) Scanning electron micrograph image of RFP-3T3 cells spreading extensively on macroporous walls in microcryogels (scale bar = 20 µm). Cytotoxicity testing of (F) doxorubicin on HepG2 cells and (I) IMMLG-8439 on NCI-H460 cells in 3D microtissue array showing increased IC50 comparing to their 2D counterparts. Data are shown as mean ± SD. (G) Small spheroids of HepG2 cells in microcryogel (scale bar = 100 µm), with (asterisks in H) partial adherence on the microcryogel wall (scale bar = 20 µm). (J) NCI-H460 cells adhered and spread within the microcryogel (scale bar = 100 µm). (K) NCI-H460 cells exhibiting fibroblastic morphology (scale bar = 20 µm). This figure has been modified with permission from reference9. Please click here to view a larger version of this figure.
Regenerative medicine and in vitro models for drug screening are two important applications for tissue engineering5,6,7,8,9. While these two applications have vastly different needs, a common ground between them lies in the need for a more biomimetic culturing condition to enhance cell functions19. Only with improved cell functions in research can we treat diseases better20,21, and if cultured cells reflect drug responses more accurately can we accelerate drug discovery6,7. Cell survival after engraftment in vivo is a crucial requirement for regenerative medicine, while throughput is important for drug screening to handle thousands of compounds at one time. These two requirements are specific to their respective applications, and seldom can one technology meet both requirements. Thus, we have uniquely integrated microfabrication technology with cryogel preparation to produce macroporous microcryogels, which could be harvested off-chip as individual cell-loaded carriers for regenerative therapy, or retained on chip for further assembly into array for high-throughput drug screening. The microscale and macroporosity of these novel microcryogels allow automatic and homogeneous loading of cells by simple absorption. Using novel microstencil fabrication of microcryogel array chips, hundreds and thousands of microscale cryogels with uniform and reproducible geometrical features could be easily and efficiently generated. The microcryogels could be prepared and stored by vacuum packaging as an off-the-shelf, ready-to-use product to facilitate preparation of 3D microtissues in common laboratories for subsequent applications. Using this fabrication technique, we were able to meet both the common ground (3D culture condition for better biomimicry) and specific requirements for the two different applications (elasticity to protect cells during injection for regenerative medicine and high-throughput in an array format for drug screening).
Cell-based therapy holds great promise for repair of various damaged tissues or organs24. However, cell retention, cell survival, and reproducibility of the treatment are still poor due to mechanical damage during injection, high leakage to surrounding tissues, and ischemia and inflammation in the in vivo environment within the lesion tissues25. Some researchers have used preformed cellular aggregates to improve free cell injection. However, it requires a large amount of cells to form cell aggregates, which leads to high cost, non-uniform size, and uncontrollable aggregate numbers26. Moreover, mechanical injury and cell death are still inevitable during injection. Alternatively, biomaterial-assisted cell therapy has been developed in which responsive biomaterials (e.g., thermal or pH-sensitive hydrogel) can be mixed with cells and gelation in situ to realize cell retention27. However, in situ cross-linked biomaterials do not allow priming of cells in vitro and result in immediate exposure of cells to an ischemic and inflammatory microenvironment at the lesion site. It is urgent to solve these problems to enhance therapeutic efficiency. A major advantage of microcryogels fabricated using this protocol is their desired injectability as a result of their miniaturized size and exceptional elasticity, facilitating their application in cell delivery. The injectability of microcryogels enables cell protection during cell delivery, hence they could be constituted into 3D injectable microtissues after in vitro priming of cells in the microcryogel to enhance both extracellular matrix (ECM) protein deposition as well as cell-cell interactions. The 3D injectable microtissues result in microscale tissue-like ensembles representing an optimal delivery strategy to facilitate cell protection, engraftment, survival, and hence improve the ultimate therapeutic effects at the lesion site.
Besides enhanced therapeutic effects for cell therapy, our results were also indicative of the complex impact of 3D microenvironment on cellular drug responses. Utilizing biomimicking culture conditions, it would be possible to elicit in vitro cellular drug responses more representative of in vivo responses, hence accelerating drug discovery6,7,28. Spheroids are popular choice of 3D cell culture configuration and many techniques have been developed to assist researchers generate spheroids. Low-adhesive tissue culture plates29 or plate surfaces that are modified with nano-imprints8 were also used to coerce cells to aggregate by preventing cell-matrix adhesion. While these techniques are relatively simple to use, problems such as loss of spheroids during medium exchange and other operations as well as size variability of spheroids are problems hindering large-scale adoption of such technology6. More homogeneous spheroids could be formed using hanging drop30,31,32,33, however it is labor intensive if not using specialized plates. Using specialized multi-well hanging drop plates, and integrating with automated liquid handling systems6,31, high-throughput screening could be realized. The largest drawback of spheroid culture is the lack of ECM, which has been identified to play vital roles in all physiological and pathological tissue developments34. A brain model study revealed that spheroids cultured inside an ECM scaffold, in comparison to pure spheroids, had increased drug resistance, enhanced acidosis due to higher lactate production and improved angiogenesis with increasing expression of related factors34. Other studies have also shown that presence of matrix could provide necessary mechano-signaling to promote EMT and supports recapitulation of tumor features such as invasion and metastasis3,35,36,37.
With increasing understanding of the importance of ECM in pathological development, there is no doubt that incorporating ECM into 3D culture methods could help mimic in vivo situations better6. Hydrogels of natural or synthetic materials have been applied to generate several in vitro 3D tumor models for assessment of chemotherapeutics due to their flexibility and controllability of biophysical properties (such as rigidity)38,39,40,41,42. While hydrogels with tunable biophysical properties had indeed modeled important biological features of tumor cells to facilitate more accurate drug screening, several disadvantages of this method have hindered its widespread use in drug screening. Crosslinking of polymers in the presence of cells is necessary to encapsulate cells within hydrogel matrix, which could potentially damage cells. Not only so, hydrogels of different biophysical properties present different challenges to cells encapsulated within. In soft hydrogels, high water content could support cell growth but such hydrogels degrade quickly, giving short-term support for 3D cell culture. On the other hand, stiffer hydrogels with high cross-linking could slow down degradation but low water content could not support cell growth and high crosslinker concentration usually induces high cytotoxicity43,44. Not only so, preparing 3D cell culture with hydrogels is labor-intensive and is not compatible with most high-throughput liquid handling systems as temperature control of hydrogel precursor solution is important and jamming of thin dispensing tips could result from gelation of hydrogel within the tips. These disadvantages have thus prompted the search for alternative ECM surrogates, i.e., scaffold-based ECM34.
Using pre-formed scaffolds, cells could be exempted from the biomaterial fabrication process and hence provide the possibility for more control over scaffold fabrication as harsher conditions could be used without fear of damaging the cells. Several investigations have shown that the tumor cells cultured in 3D scaffolds display higher drug resistance compared with cells cultured in 2D due to increased malignancy and enhanced cell-ECM interaction45,46,47. Such observations are consistent with our results presented here. In our other works, we have further demonstrated the versatility of the 3D microtissue array and its advantages over the other techniques mentioned above. In a recent work, we were able to enhance hepatic function by promoting the epithelial phenotype of HepaRG cells by culturing on a 3D microtissue array and such array was applied to drug hepatotoxicity evaluation47. Owing to uniformity of pore sizes within the 3D macroporous scaffolds, we were able to control the size of liver cell spheroids to fall within 50-80 µm, regardless of the initial cell-seeding density. This provides a significant advantage over free-forming spheroids with non-uniform sizes. Not only do spheroids grow uniformly within each scaffold, cells between wells are also uniformly seeded, giving Coefficient of Variation (CV) comparable with cells seeding in 2D (i.e., CV = 0.09 in 3D microtissue array and CV = 0.05 in 2D commercial plate; data not shown). In another work, we have demonstrated that we were able to form liver microtumor on 3D microtissue array to recapitulate tumor-stromal interactions for screening of stroma-reprogrammed combinatorial therapy48. Liver microtumors were generated by long-term (5 days) co-culture of fibroblasts with tumor cells at high density. We observed barriers towards drug diffusion due to the compact cell and ECM structure formed in the liver microtumor, which was similarly observed in vivo32. Using luciferase-labeled cancer cells and mechanically-primed stromal cells, combined with luminescence of luciferin as the specific read-out for cancer cells, high-throughput screening of novel therapeutic agents or combinations against tumor-stromal interaction is made possible.
Despite the many unique advantages of our technique, a drawback of current microcryogels is non-transparency, hindering detailed optical observation of cells in microcryogels. Further improvements for these microcryogels would include fine-tuning their optical properties to enhance imaging of cells in microcryogels for observation. Also, important biophysical properties such as rigidity have not been explored in our technique, which would need to be addressed if we want to better mimic physiological and pathological tissues of different biophysical properties.
While our technique allows for simple generation of 3D microtissues, some cautions must be taken for successful experiments. When fabricating 3D microcryogels on microstencil array chip, it is important to ensure that the microcryogels remain frozen when placed in the lyophilizer. Hence it is essential to pre-cool the lyophilizer and to transfer microcryogels from the -20 °C freezer to the lyophilizer quickly. The melting of microcryogels before lyophilizing or during lyophilizing will cause pores to collapse and hence affect the porosity of microcryogels fabricated. When culturing 3D microtissues in the array format, attention is required to ensure the adhesiveness between the two arrays is sufficient to prevent cross-contamination between wells. Also, while miniaturizing cell culture has its advantage in increasing the throughput and reducing reagent consumption, its drawback is that the low culture volume could not support long-term cell culture without frequent media replenishment. Not only so, it is critical to maintain the humidity of the culture environment to prevent influence on cell viability due to media evaporation, since only a few microliters of cell suspension or media is added. Evaporation will also affect the viability of cells in the peripheral wells, hence it is vital to avoid culturing cells in these wells.
Nonetheless, our robust technique provides an option to generate 3D microtissues in an easy-to-use manner, which could potentially make 3D culture a common cell manipulation method for most laboratories, to accelerate both basic and translational science advancements.
The authors have nothing to disclose.
This work was financially supported by the National Natural Science Foundation of China (Grants: 81522022, 51461165302). The authors would like to acknowledge all Du lab members for general assistance.
Gelatin | sigma | G7041 | All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. |
Glutaraldehyde | J&K | 902042 | Used as crosslinker in preparation of material. |
Glass cover slip (24X50mm) | CITOGLASS, China | 10212450C | To scrape prcursor solution onto microstencils array chips. |
Sodium borohydride, NaBH4 | Beijing Chemical Works | 116-8 | To wash remaining glutaraldehyde away after gelation. |
Vacuum jar | asperts, China | VC8130 | To preserve microgels under vacuum. |
Polymethylmethacrylate (PMMA) sheets | Sunjin Electronics Co., Ltd, China | Ordinary PMMA sheets. | |
Rayjet laser system | Rayjet, Australia | Rayjet 50 C30 | To engrave PMMA sheets to form wells. |
Plasma Cleaner | Mycro Technologies, USA | PDC-32G | To make PMMA hyphophilic. |
Lyophilizer | Boyikang, China | SC21CL | To lyophilize materials. |
Trypan Blue solution (0.4%) | Zhongkekeao, China | DA0065 | To dye microgels. |
Doxorubicin hydrochloride | ENERGY CHEMICAL, China | A01E0801360010 | To test drug resistance of cells in 2D or 3D microgel. |
Live/dead assay | Dojindo Molecular Technologies (Kumamoto, Japan) | CS01-10 | To distinguish alive and dead cells. |
Cell Titer-Blue | Promega (Wisconsin, USA). | G8080 | To test cell viability. |
Cell strainer | BD Biosciences, USA | 352360 | To collect microgels. |
D-Luciferin | SYNCHEM (Germany) | s039 | To tack cells. |
Scanning electron microscope | FEI, USA | Quanta 200 | To characterize microgel morphology. |
Mechanical testing machine | Bose, USA | 3230 | To measure mechanical features. |
Programmable syringe pump | World Precision Instruments, USA | ALADINI 1000 | To test injactabiliy. |
Digital force gauge | HBO, Yueqing Haibao Instrument Co., Ltd., China | H-50 | To test injactabiliy. |
Ethylene oxide sterilization system | Anprolene, Anderson Sterilization, Inc., Haw River, NC | AN74i | To sterilize microgels with ethylene oxide gas. |
Microplate reader | Molecular Devices,USA | M5 | To measure fluorescence intensity in micro-array. |
Confocal microscope | Nikon, Japan | A1Rsi | To observe cell distribution in 3D. |
Xenogen Lumina II imaging system | Caliper Life Sciences, USA | IVIS | To track cell in animals. |
Liquid work stataion | Apricot design,USA | S-pipette | To load medium or cell suspension high-throuputly. |