1.Background of Laminin 521
Laminin 521 (LN521) is a key cell adhesion protein in the natural stem cell niche and a matrix for the culture and expansion of human embryonic (hES) and induced pluripotent stem cells (hiPSC). LN521 binds to cell surface receptors and activates cell signaling pathways, resulting in the production of more functional cells.
Laminin 521 reproduces the biologically relevant hPSC environment in vitro, promotes the attachment, high survival and strong long-term self-renewal of hPSC, and is a key matrix protein that supports stem cell growth and maintains the stability of basement membrane structure and performance. Laminin 521 is also one of the most commonly used nutrient-free culture matrices. In addition, Laminin 521 can achieve efficient single-cell passaging of genetically stable and pluripotent stem cells without the addition of any apoptosis inhibitors. The cells grow in a uniform monolayer without the need to manually remove any differentiation areas. In addition, studies have shown that LN521 can support PSC growth for more than 10 generations without any signs of karyotype abnormalities, and maintains the ability of PSC to differentiate into the three germ layers of the inner, middle and outer germ layers.
Figure 1. Laminin 521 structure [1]
2.Laminin coating experiment
2.1 Prepare laminin 521 to 400µg/ml with sterile deionized water or water for injection. It is recommended to further dilute to 100µg/ml with sterile 1×DPBS (Ca++/Mg++) before use, and then dilute to a working concentration of 5-10µg/ml with sterile DPBS (Ca++/Mg++). The coating concentration varies depending on the type of cultured cells, and it is recommended to optimize the experimental protocol to determine the optimal coating concentration for the cells. Refer to Table 1 for recommended concentrations.
Note: DPBS containing Ca2+ and Mg2+ should be used, as divalent cations are important for protein structure and function.The required working concentration of Laminin 521 depends on the cells and the application. We recommend an initial coating concentration of 0.5μg/cm2.
Table 1. Recommended volumes of recombinant human laminin working solution for different culture vessels.
|
Petri dish |
Coating concentration（μg/mL） |
Addition amount of LN521 |
Addition amount of 1*DPBS |
Total volume |
|
6 well |
5 |
50 μL/well |
950 μL/well |
1 mL/well |
|
12 well |
5 |
25 μL/well |
475 μL/well |
0.5 mL/well |
|
24 well |
5 |
15 μL/well |
285 μL/well |
0.3 mL/well |
|
48 well |
5 |
7.5 μL/well |
142.5 μL/well |
150 μL/well |
|
96 well |
5 |
3.5 μL/well |
66.5 μL/well |
70 μL/well |
|
35mm Petri dish |
5 |
50 μL/well |
950 μL/well |
1 mL/well |
|
60mm Petri dish |
5 |
100 μL/well |
1900 μL/well |
2 mL/well |
|
100mm Petri dish |
5 |
300 μL/well |
5700 μL/well |
6 mL/well |
The above volume is based on a coating concentration of 5μg/mL.
2.2 Add the specified volume of laminin-DPBS mixture to each well and shake gently. Make sure the entire surface is covered with laminin coating solution, as uncoated surfaces do not support cell growth.2.3. Place the plate in a 37°C incubator and incubate overnight. The minimum incubation time is 2 hours, but overnight incubation is recommended to obtain ideal cell culture conditions. Be careful not to let the culture container dry out, which will inactivate LN521.
2.4. When the cells are ready to be seeded, aspirate the laminin 521 solution.
3.iPSC Culture
3.1 iPSC thawing (taking 12-well plate as an example)
3.1.1 Remove iPSCs from liquid nitrogen or dry ice and thaw in 37°C water for 10 seconds.
3.1.2 Sterilize the cryovials with 75% alcohol and transfer them to a work surface.
3.1.3 Transfer the cells to a new 15 mL centrifuge tube containing 9 mL DMEM‑F12.
3.1.4 Centrifuge the 15 mL centrifuge tube at 300g for 5 minutes at room temperature.
3.1.5 Discard the laminin solution from the coated 12-well plate.
3.1.6 Discard the cell supernatant and gently resuspend the iPSCs in 1 mL mTeSR-plus (containing 10 µM Y-27632) and transfer them to the coated 12-well plate. Shake the plate to evenly distribute the cells to a final cell density of 1×10^5 cells/well and let stand at room temperature for 10 minutes. Ensure that the culture is passaged within 4-5 days.
3.1.7 Return the 12-well plate to the 37°C incubator for incubation.
3.1.8 The culture medium containing ROCK inhibitor should be removed after 12-16 hours and continue to be cultured in the medium without inhibitor.
Table 2. Cell seeding density in different culture vessels, cell number determined according to cell growth rate
|
Cell culture vessels |
6 well |
12 well |
24 well |
96 well |
|
Cell number |
2.5~3.5×105 |
6~8×104 |
3~4×104 |
0.5~1×104 |
3.2. iPSC Passaging Protocol
3.2.1 Discard the culture supernatant and rinse with 1 mL PBS (Ca‑‑/Mg‑‑).
Note: Use PBS without Ca2+ and Mg2+, as divalent cations have a negative effect on some dissociation enzymes.
3.2.2 Discard the PBS and add 0.5 mL Gentle Cell Dissociation Reagent. Incubate at room temperature for 6-8 minutes or observe under a microscope until the cells are no longer bound to the plate.
3.2.3 Add an equal volume of DMEM-F12, gently mix the cells, transfer to a centrifuge tube at room temperature, and centrifuge at 300g for 5 minutes.
3.2.4 Before passaging cells, prepare a laminin-coated 12-well plate.
3.2.5 Discard the supernatant and resuspend the cells in mTeSR‑plus medium containing 10 µM Y‑27632.
3.2.6 Transfer the cells to the prepared laminin-coated 12-well plate. Gently shake the plate to evenly distribute the cells and leave at room temperature for 10 to 20 minutes.
3.2.7 Place the 12-well plate in a 37°C incubator and incubate.
Note: iPSCs will rapidly differentiate and die after growing into a monolayer. To maintain growth and pluripotency, they need to be passaged before they are confluent.
3.3. Cryopreservation of iPSC
3.3.1 Prepare the gentle cell dissociation reagent.
3.3.2 Perform cell separation according to the iPSC passaging protocol, use the cell organelle count to ensure the cell density before cryopreservation.
3.3.3 Each tube of cells should be frozen at a cell density of 1-2×10^6. Follow the steps of centrifugation and removal of cell culture medium in the iPSC passaging protocol. Resuspend the isolated iPSC pellet in an appropriate volume of cryopreservation solution
3.3.4 Add 1 mL of resuspended cell cryopreservation pellet to a 1.5 mL cryopreservation tube, perform program cooling, and then transfer to liquid nitrogen for long-term storage.
4.Important Notes on Laminin Coating
4.1. All steps in the experimental protocol must be performed under sterile conditions.
4.2. Avoid exposing laminin to room temperature for a long time.
4.3. Avoid repeated freezing and thawing
4.4. After thawing, the undiluted protein stock solution can be stored at 2~8℃ under sterile conditions and can be stored stably for at least 3 months.
5.Related product information
|
Product Name |
Cat# |
Size |
|
Recombinant Human Laminin 521 Protein（Animal-Free） |
92602ES |
10μg/100μg/500μg/1mg |
6.References
[1]Pulido D, Briggs DC, Hua J, Hohenester E. Crystallographic analysis of the laminin β2 short arm reveals how the LF domain is inserted into a regular array of LE domains. Matrix Biol. 2017 Jan;57-58:204-212.