实验概要

The  AlgiMatrix™ 3D Culture System is an animal origin-free bioscaffold that  facilitates three-dimensional (3D) cell culture. Each bioscaffold is an  alginate sponge with a pore size of ~50–200 μm. Cells grown in this  sponge more closely match normal cell morphology and behavior, providing  an excellent solution for 3D cell-culture models in many research  fields, such as toxicology, drug development, cancer research, and  tissue engineering.

AlgiMatrix™  bioscaffolds are supplied lyophilized in sterile plate wells and are  stable at room temperature. The AlgiMatrix™ 3D Culture System is  suitable for many cell-based screening and drug discovery procedures,  including Multicellular Tumor Spheroid Assays (MCTS), hepatocyte and  cardiomyocyte organogenesis studies, co-culture studies, high-throughput  drug screening, and embryonic stem-cell 3D differentiation studies.

实验步骤

1.         Resuspend the cells in standard cell culture medium, and then add 10%  (v/v) AlgiMatrix™ Firming Buffer to this suspension (i.e., 1 part  Firming Buffer to 9 parts cells plus medium). The optimal final cell  concentration will vary by cell type, but in general 1 × 10⁶  cells/mL is a reliable target. While 10% (v/v) Firming Buffer provides  an optimal balance between sponge transparency and firmness, certain  applications may benefit from optimizing this concentration.

2.        Remove the AlgiMatrix™ 3D Culture System plate from its package and discard the desiccant.

3.         Depending on your plate type, inoculate the following volume of the  suspension from Step 1 onto the top of each dry sponge with a pipette:

6-well Plate            24-well Plate           96-well Plate

2 mL                     400–500 μL            80–120 μL

4.         Optional:  Dynamically seed the sponges by immediately centrifuging the  plates at 100 × g for 4 minutes. Certain cell types may be embedded  more thoroughly within the sponge with dynamic seeding.

Note:  The sponge will become wet and translucent. Gas bubbles may appear in  the matrix; they will decrease or disappear with time. If large bubbles  appear inside or under the sponge, release by gently pushing the sponge  against the plate bottom with pipette tip.

5.         Approximately 5 minutes after rehydration, the sponge should appear  foamy with a dry or soggy surface. Depending on your plate and cell  type, add the following additional volumes of culture medium without  Firming Buffer to the top of each sponge (volumes are intended as a  general starting point; volumes may vary by cell type):

6-well Plate           24-well Plate           96-well Plate

2–3 mL                 400–500 μL            80–120 μL

6.        Incubate the plate(s) in an incubator (36–38°C in a humidified atmosphere of 4–6% CO₂ in air). Do not stack multiple plates.

7.         Change the medium based on cell proliferation or when the medium begins  to change color. To observe the cells inside or on top of the  AlgiMatrix™ sponge, decrease the medium volume or relocate the sponge to  a dry well. Sponges can be inverted for better observation of cells at  the top of the sponges.

Note:  Do not allow the aspirating pipette tip to contact the sponge when  removing spent medium. Keep the tip on an angle against the wall of the  well and block the tip with another pipette tip to avoid sucking up the  sponge (see image). If the sponge becomes stuck in the aspirator, stop  suction right away and release the sponge, then gently try again. If the  sponge has been partially aspirated, do not use that well for  quantitative assays. Refer to Tips at the top of the page for additional  suggestions regarding medium exchange.

 Picture 1

8.         After several days, remove the plate from the incubator and examine  under light microscopy (low magnification) for the presence of spheroid  formation.

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