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Assembling 3D Devices With Photos (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Assembling 3D Devices With Photos (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Racheal Jappert; Jeffrey Pippin; Ying Zheng; Stuart Shankland

Keywords

[‘3D culture’]

Subjects

[‘Cell culture’, ‘Immunological techniques’]

Release Date

2017-05-26

Abstract

Describes the procedures for casting and seeding cells into a three-dimensional microvascular culture system.

Equipment

Washed and Autoclaved Equipment Needed

  • 2 sets of tweezers
  • Stainless steel spatula
  • Stainless steel screws
  • Stainless steel dowel pins
  • Micropatterned PDMS molds
  • PDMS flat squares
  • Glass coverslips (22x22mm)
  • Cotton
  • Top and bottom PMMA pieces

Procedure

Reconstituting Lyophilized Collagen

  1. Take tube of lyophilized collagen (looks like cotton) out of freezer and keep on ice.
  2. Add appropriate amount of cold 0.1% acetic acid to the collagen to reconstitute at 15mg/mL and place tube at 4°C. For example, if the lyophilized collagen (excluding weight of tube) weighs 225mg, then add 15mL of cold 0.1% acetic acid (225mg/15mL = 15mg/mL). It will take ~4 days for the lyophilized collagen to completely dissolve.
  3. Mix the collagen as needed (about once per day) in a sterile environment until fully reconstituted. The collagen is fully dissolved when there are no longer visible fibrils/strings present. The reconstituted collagen should be a viscous, clear liquid with no visible collagen fibrils.
  4. Spin collagen at 4°C, 2000 rpm (1000G) for 8 minutes to remove any air bubbles that may have been introduced during this process.

Preparing Collagen Gel

  1. Add appropriate amounts of sodium hydroxide (NaOH), 10x Media Supplement (M199), and cell culture media to a 30mL conical tube on ice and mix thoroughly with a small spatula.
  2. Add collagen, mixing until a homogeneous gel is obtained. Mixture should be pinkish in color.
  3. If needed, centrifuge at 1000G for 8 minutes to remove air bubbles.
  4. To incorporate cells, spin down appropriate number of cells to achieve desired concentration (up to 0.5 million cells/mL) in 30mL conical tube. After aspirating off liquid, add collagen mixture to cells and gently mix with spatula to evenly distribute cells. If bubbles become introduced, spin again at 1000G for 8 minutes and mix gently to re-suspend cells in mixture.

Collagen Injection – Top Piece

  1. Place micropatterned Polydimethylsioxane (PDMS) mold in a 100mm x 20 mm dish and plasma treat PDMS mold for 1 minute.
  2. Align top PMMA piece on top of the PDMS mold so that the square reservoirs on the mold are directly under the inlet and outlet reservoirs.
  3. Place stainless steel dowel pins into the inlet and outlet reservoir holes on the top PMMA piece to maintain clear access from the reservoirs to the pattern.
  4. If the PMMA piece has extra holes besides the inlet and outlet, place a screw head down within the hole to prevent gel from passing through.
  5. Use a 1mL syringe to extract ~0.6mL collagen. Ensure that no bubbles remain in the syringe before injecting into top piece.
  6. Press down lightly with tweezers on the top housing to ensure flush contact between the top piece and the PDMS mold.
  7. Inject collagen slowly through an injection port on the top PMMA piece. Check that collagen fills in the 20 mm x 20 mm domain above the pattern and does not leak out. NO BUBBLES.
  8. Close the dish without jostling the dowel pins and allow to gel at 37°C for 30 minutes.

FIGURE 1: Visual instructions for Top Piece Collagen injection and gelation

Collagen Injection – Bottom Piece

  1. Place a 22x22 mm glass coverslip in the center of the bottom piece.
  2. Use a1 mL syringe to dispense ~0.25 mL collagen onto the glass slide. Add slightly more gel to the side of the device where the stamp will be laid down first.
  3. Gently lower the PDMS flat square over the collagen ensuring the PDMS is flat against the PMMA and there are no trapped bubbles. Using the “stickier” side of the stamp prevents sliding.
  4. Allow to gel at 37°C for 30 minutes.

FIGURE 2: Bottom piece Part 1

FIGURE 3: Bottom piece Part 2

Device Assembly

  1. After gelation, use tweezers to remove excess collagen around the edges of the PDMS square on the bottom piece.
  2. Add RPMI around the edges of the flat PDMS and slowly peel off the PDMS piece.
  3. Add more RPMI on top of the collagen to maintain hydration.
  4. Remove the top piece and stamp from the bottom of the petri dish. This can be challenging: a. Use RPMI to separate the stamp from the bottom of the dish. b. Firmly grasp device with tweezers and flip over the petri dish and slowly pull the dish away from the stamp. c. Add RPMI around the stamp and then quickly and firmly remove the PDMS mold from the top PMMA piece. d. Cover the top piece in RPMI to prevent drying out. Air contact can cause disruptions in the gel, causing devices to not fuse properly and be leaky.
  5. Gently remove stainless steel dowel pins from the PMMA housing using another pair of tweezers.
  6. Using a 200ul pipette tip, ensure the inlet and outlet openings are not blocked by gel.
  7. Flip the PMMA top piece over so that the collagen faces down and add place 3 screws into the corner holes. a. Gently line up the left side screw holes and begin slowly laying down the top piece on the bottom piece from left to right using a spatula as a lever to prevent sliding or dropping the pieces together. This prevents the top channels from being crushed. b. Ensure the right screw holes are lined up before removing the spatula.
  8. Add the fourth screw into the remaining hole and use a spatula to tighten the screws. Do not under or over tighten. Do it just right.
  9. Aspirate the RPMI surrounding the PMMA surfaces and place a small piece of cotton under one edge of the device.
  10. Using a pipette, remove any RPMI from the reservoirs and replace with cell culture media.
  11. Allow the device to gel together in a 37°C incubator for 1-3 hours.

Cell Seeding

  1. After acutasing cells, run through a 100um cell strainer. Add equal amounts of appropriate media through the strainer.
  2. Spin cells at 1000 g for 5 minutes.
  3. Resuspend cells in 1 mL media and do a cell count to determine the number of cells present.
  4. Resuspend the cells in the appropriate amount of media to achieve desired cell concentration:
    • 5-10 million cells/mL when seeding the lumen
    • up to 0.5 million cells/mL when seeding matrix
  5. Remove all cell culture media from the inlet and outlet reservoirs.
  6. Using a 200ul gel loading tip, add 10ul of cell suspension into the center of the inlet reservoir. The cells should begin to flow into the network immediately and coat the network.
  7. Add 200ul medial equally to both reservoirs and let cells attach and spread within the network for at least 1 hour.

Consortium

(Re)Building a Kidney (RBK) Consortium