fabrication of square pyramidal microwells for tumor spheroid fabrication

PEG 하이드로겔에서 종양 스페로이드의 3D 캡슐화 후

Tumor spheroids have emerged as useful in vitro tools in studying cancer etiology, pathology, and drug responsiveness1. Traditionally, spheroids have been cultured in conditions such as low adhesion plates or bioreactors, where cell-cell adhesion is favored over cell-surface adhesion2. However, it is now recognized that to recapitulate the tumor microenvironment more faithfully, in vitro spheroid models should capture both cell-cell and cell-matrix interactions. This has prompted multiple groups to design scaffolds, such as hydrogels, where spheroids can be encapsulated3,4. Such hydrogel-based spheroid models enable the elucidation of cell-cell and cell-matrix interactions on various cell behaviors, such as viability, proliferation, stemness, or therapy responsiveness3.
Here, we describe a protocol for the encapsulation of glioblastoma spheroids in polyethylene glycol (PEG) hydrogels. There are multiple literature reports of glioblastoma cell spheroid encapsulation in hydrogels. For example, spheroids were formed by encapsulating U87 cells in PEG hydrogels decorated with an RGDS adhesive ligand and crosslinked with an enzymatically cleavable peptide to determine the effect of hydrogel stiffness on cell behavior5. U87 cells have also been formed in other PEG-based or hyaluronic acid-based hydrogels to expand the cancer stem cell population6 or to explore matrix-mediated mechanisms of chemotherapy resistance7,8,9. Glioblastoma spheroids have also been encapsulated in gelatin hydrogels to study the crosstalk between microglia and cancer cells and its effect on cell invasion10. Overall, such studies have demonstrated the utility of hydrogel-based in vitro models in understanding glioblastoma pathology and devising treatments.
Further, there are different methods for tumor spheroid fabrication and hydrogel encapsulation11. For example, dispersed cells could be seeded in hydrogels and allowed to form spheroids over time5,12. One drawback of such a method is the polydispersity of the formed spheroids, which could lead to differential cell responses. To produce uniform spheroids, cells could be encapsulated in microgels and cultured for extended periods until they invade and remodel the gel13, or cells could be deposited in templated gels with spherical &#39;holes&#39; and allowed to aggregate14. The drawback of these methods is their relative complexity, the need for a droplet generator or other means to form microgels or the &#39;holes&#39; in the gel, and the time it takes for spheroids to grow and mature. Alternatively, spheroids could be pre-formed in microwells9,15,16 or in hanging-drop plates17,18 and then encapsulated in a hydrogel, similar to the technique described here. These methods are simpler and can be done in a higher throughput fashion. Interestingly, it has been shown that the method of spheroid formation can affect spheroid cell behaviors, such as gene expression, cell proliferation, or drug responsiveness19,20.
Here, we focus on glioblastoma since it is a solid tumor whose native environment is the soft, nanoporous brain matrix21, which can be mimicked by a soft, nanoporous hydrogel. Glioblastoma is also the deadliest brain cancer for which there is no available cure22. However, the protocol described here can be used for the encapsulation of spheroids representing any solid tumor. We chose to use PEG hydrogels that are formed through a Michael-type addition reaction23. PEG is a synthetic, non-degradable, and biocompatible hydrogel that is inert and serves as scaffolding and physical cell support but does not support cell attachment23. Cell adhesiveness can be added separately via tethering of whole proteins or adhesive ligands24, and degradability can be added via chemical modifications of the PEG polymer chain or hydrolytically or enzymatically degradable crosslinkers25,26. This allows for biochemical properties to be tuned independently of mechanical or physical hydrogel properties, which could be advantageous in studying cell-matrix interactions. The Michael-type gelation chemistry is selective and happens at physiological conditions; hence, it allows for spheroid encapsulation by simply mixing the spheroids with the hydrogel precursor solution.
Overall, the methodology presented here has several notable characteristics. First, fabricating tumor spheroids in a multiwell assembly is efficient, quick, and the cost of the required materials is low. Second, the spheroids are produced in large batches in a variety of sizes with low polydispersity. Finally, only commercially available materials are required. The utility of the methodology is illustrated by exploring the effect of substrate properties on spheroid cell viability, circularity, and cell stemness.

저자 스포트라이트: 교모세포종 미세환경 연구를 위한 체외 하이드로겔 모델

스페로이드-매트릭스 상호 작용 연구를 위한 폴리에틸렌 글리콜 하이드로겔의 종양 스페로이드 제조 및 캡슐화

Our lab focuses on designing in vitro hydrogel models for glioblastoma steroid encapsulation to mimic the tumor extracellular microenvironment. The effective substrate, mechanical and biochemical properties on steroid cell viability, circularity, stemness, and spreading are investigated with such models. Current experimental challenges in tumor module development revolve around a desire for complexity to mimic all aspects of the tumor microenvironment for physiological relevance, and also a desire for simplicity to make the model accessible, robust, low cost, and easy to use, and compatible with multiplex screening approaches.We present a protocol that enables fast, robust, and low-cost fabrication of tumor steroids and encapsulation in hydrogels made to mimic the tumor microenvironment. The model does not require any specialized equipment. It will be particularly useful for exploring steroid matrix interactions in building in vitro tissue physiology or pathology models.Understanding the interactions between glioblastoma cancer cells and their microenvironment can encourage development of new therapies to better treat this cancer. Furthermore, development of an in vitro model for glioblastoma cancer can enhance development of this model for other cancer diseases.

종양 스페로이드 제작을 위한 정사각형 피라미드형 마이크로웰 제작

시작하려면 2g 또는 약 1밀리리터의 음성 PDMS 전구체 용액을 6웰 정사각형 파라메탈 마스터 몰드의 한 웰에 붓습니다. 마스터 몰드를 PDMS로 덮은 후 플레이트를 진공 건조에 넣어 용액의 가스를 제거합니다. 그런 다음 접시를 섭씨 60도의 오븐에 24시간 동안 넣어 PDMS를 경화시킵니다.PDMS가 경화되었지만 여전히 따뜻하면 주걱을 사용하여 마스터 금형에서 PDMS 금형을 조심스럽게 제거합니다. 그런 다음 생검 펀치로 직경 35mm의 슬래브로 자릅니다. 적절하게 절단된 PDMS 몰드를 페트리 접시에 넣고 뚜껑으로 덮어 실온에서 추가로 24시간 동안 계속 경화시킵니다.포지티브 PDMS 몰드를 준비하려면 네거티브 PDMS 몰드의 35mm 슬래브를 질감이 있는 마이크로 웰이 위를 향하도록 하여 35mm 페트리 접시에 놓습니다. 2.5g 또는 약 1.2ml의 양극 PDMS 전구체 용액을 페트리 접시의 음극 몰드에 붓습니다. 전구체 용액을 30분 동안 가스를 제거한 후 섭씨 60도의 오븐에 3-4시간 동안 넣습니다.경화 후 페트리 접시에서 곰팡이를 제거합니다. 즉시 네거티브 몰드에서 포지티브 몰드를 벗겨냅니다. 10mm 생검 펀치를 사용하여 포지티브 몰드를 슬래브로 자릅니다.그런 다음 핀셋을 사용하여 각 10mm 포지티브 몰드의 평평한 면을 미리 준비된 PDMS 접착제 전구체 용액에 부드럽게 담그십시오. 48웰 플레이트의 웰에 웰당 하나의 몰드를 조심스럽게 놓습니다. 각 금형을 부드럽게 눌러 우물 바닥에 붙입니다.조립된 플레이트를 섭씨 60도에서 4-24시간 동안 배양하여 접착제를 경화시킵니다.

fabrication-square-pyramidal-microwells-for-tumor-spheroid

Research

JoVE Journal

Bioengineering

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

70 조회수.  Saint Louis University. 시작하려면 2g 또는 약 1밀리리터의 음성 PDMS 전구체 용액을 6웰 정사각형 파라메탈 마스터 몰드의 한 웰에 붓습니다. 마스터 몰드를 PDMS로 덮은 후 플레이트를 진공 건조에 넣어 용액의 가스를 제거합니다. 그런 다음 접시를 섭씨 60도의 오븐에 24시간 동안 넣어 PDMS를 경화시킵니다.PDMS가 경화되었지만 여전히 따뜻하면 주걱을 사용하여 마스터 금형에서 PDMS 금형을 조심?...

비디오: 종양 스페로이드 제작을 위한 정사각형 피라미드형 마이크로웰 제작

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

연구

생체공학

To begin pour two grams or about one milliliter of negative PDMS precursor solution onto one well of a six well square parametal master mold. After covering the master mold with PDMS, place the plate in a vacuum desiccate to degas the solution. Next, place the plate in an oven at 60 degrees Celsius for 24 hours to cure the PDMS.Once PDMS cures but is still warm, use a spatula to carefully remove the PDMS mold from the master mold. Then with a biopsy punch, cut it into a 35 millimeter diameter slab. Place the properly cut PDMS mold in a Petri dish and cover it with the lid to continue curing it at room temperature for an additional 24 hours.To prepare the positive PDMS mold place a 35 millimeter slab of the negative PDMS mold in a 35 millimeter Petri dish with the textured micro wells facing up. Pour 2.5 grams or about 1.2 milliliters of positive PDMS precursor solution onto the negative mold in the Petri dish. Degas the precursor solution for 30 minutes before placing it into the oven at 60 degrees Celsius for three to four hours.After curing, remove the molds from the Petri dish. Immediately peel the positive mold from the negative mold. Use a 10 millimeter biopsy punch to cut the positive molds into slabs.Next, use tweezers to gently dip the flat side of each 10 millimeter positive mold into the pre-prepared PDMS glue precursor solution. Carefully place one mold per well in a well of a 48 well plate. Gently press each mold to glue it to the well bottom.Incubate the assembled plate at 60 degrees Celsius for four to 24 hours to cure the glue.

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

To begin, place the PDMS microwell molds, anti-adherence rinsing solution and necessary lab wear in a tissue culture hood. To wash the PDMS microwell molds, use a 1, 000 microliter pipette to add 300 microliters of anti-adherence rinsing solution to each well. Then centrifuge the plate, 1, 620 G for three minutes.Use a vacuum pump and a Pasteur pipette to aspirate the solution. Next, place 500 microliters of cell suspension at the desired concentration in the microwells and centrifuge the plate at 1, 620 G for three minutes. Place the plate in a humidified incubator to allow spheroid formation.To harvest the spheroids, using a 1, 000 microliter pipette, firmly add 500 microliters of complete medium into each well. Pipette the added medium up and down at each quadrant three to four times to dislodge the speroids. Then use the pipette to gently aspirate the medium containing the spheroids into a microcentrifuge tube.Transfer 50 microliters of the steroid suspension in a microcentrifuge tube. Then sequentially add 4-Arm PEG acrylate and PEG dithiol into it. Mix the resultant solution by pipetting it up and down about 10 times.To yield 100 microliters of a 10%weight by volume PEG hydrogel precursor solution, pipette 20 microliters of the gel precursor solution in between two parafilm-lined glass slides separated with one millimeter silicon spacers and incubate the slides with gel precursor solution to allow for gelation. Once hydrogel gelation is complete, using a spatula, gently peel the gels off the separated glass slides. Place one gel per well into a 24-well plate, ensuring the surface containing the spheroids faces up.Add 500 microliters of complete medium to each well to submerge the hydrogel completely. Place the multi-well plate in a humidified incubator to culture the cells. The described technique using microwell molds resulted in the formation of spheroids with spherical shapes and tightly-controlled polydispersity.For spheroids with 3, 300 cells per microwell, the average spheroid size was about 250 microns and circularity was greater than 0.8, considering a value of 1 as a perfect sphere. Spheroid diameters were dependent on the microwell size and the number of cells per microwell.

하이드로겔의 다세포 종양 스페로이드 형성, 수확 및 캡슐화

Immunofluorescence Fixation, Staining, and Clearing of Encapsulated Spheroids for Confocal Microscopy

To begin, aspirate the media from the wells where the hydrogel-encapsulated cells are cultured in the 24-well plate. Rinse the hydrogels by pipetting 500 microliters of PBS directly onto each well containing the hydrogels, and then gently aspirate the PBS. Using a 1, 000 microliter pipette, add 500 microliters of fixative solution per well to fix the spheroids in the 24-well plate.Allow the fixative to soak the gels for 30 minutes at room temperature, before pipetting out the fixative solution and discarding it in a designated waste container. Next, rinse the hydrogels with PBS three times as demonstrated previously. If not used immediately, store the hydrogels in 500 microliters of PBS per well at four degrees Celsius for up to one week.To stain in the hydrogel-encapsulated cells, first, prepare appropriately diluted primary antibodies for nestin and SOX2. After aspirating the PBS from the wells, add 50 microliters of the diluted antibodies to each well. Incubate the cells with the antibodies for 24 hours to stain them completely.Then using a 1, 000 microliter pipette, remove the staining solution and discard the waste appropriately. After rinsing the hydrogels three times, store them in PBS at four degrees Celsius for up to two weeks prior to imaging, or image immediately. To clear the stained spheroid for improving imaging transparency, first aspirate the PBS from each well.Then sequentially treat the spheroids with 500 microliters of 20%40%and 80%formamide per well for 90 minutes each. Finally incubate the hydrogels in 100%formamide for 24 hours prior to imaging. The spheroids were imaged at varying z-stack depths, allowing for the cell viability assessment at each location within the z-stack.A maximum projection utilizing the spheroid stack represented the highest point of light intensity within each location, simplified into one image. Representative images of stained spheroids revealed that the self-renewal transcription factor, SOX2, was co-localized with DAPI in the nucleus. On the contrary, stem cell marker nestin was present throughout the cells.There was no difference between the free floating spheroid without gel and the encapsulated spheroids, possibly due to the inertness of the PEG gel used. The representative images of optical sections at about 90 microns into cleared and uncleared spheroids revealed that when clearing was performed, the spheroid core could be imaged about 30 microns deeper compared to an uncleared spheroid.