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User Manual

VIEWS: 13 PAGES: 20

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User Manual

OriCellTM Strain C57BL/6 Mouse
Adipose-derived Mesenchymal
Stem Cells (ADSCs)
Cat No. MUBMD-01001
    Table of Contents

    Contents and Storage ……………………………………………………………………………………… 3

    Product Introduction   …………………………………………………………………………………… 3

    Cell Characteristics and Identity ……………………………………………………………………… 3

    Product Applications ……………………………………………………………………………………… 3

    Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs Source ………………………………………… 4

    General Handling Principles …………………………………………………………………………… 4

    Preparing Growth Medium …………………………………………………… 4
    Preparing OriCellTM ADSC Growth Medium ………………………………………………………… 4

    Culturing OriCellTM Strain C57BL/6 Mouse ADSCs …………………… 6
    Thawing and Establishing OriCellTM Strain C57BL/6 Mouse ADSCs ………………………… 6

    Passaging Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs …………………………………… 7

    Differentiaon of OriCellTM Strain C57BL/6 Mouse ADSCs ……………………………………… 9

    Cryopreservation of OriCellTM C57BL/6 Mouse ADSCs ………………………………………… 17

    Appendix ………………………………………………………………………………… 19

    Trouble Shooting …………………………………………………………………………………………… 19

    Related Products …………………………………………………………………………………………… 20




2
Contents and Storage

                                    Strain C57BL/6 Mouse Adipose-derived
             Product Name
                                    Mesenchymal Stem Cells

               Catalog No.          MUBMD-01001


                   Vials            1


            Amount per Vial         1×106 Cells


                                    Liquid Nitrogen

       CAUTION: Handle this as potentially biohazardous material for it contains Dimethyl
       Sulfoxide (DMSO), a hazardous material, in the freezing medium.



Product Introduction
       Adipose-derived Mesenchymal Stem Cells (ADSCs) are multipotent stem cells that can
       differentiate into a variety of cell types, including osteocytes, adipocytes and chondrocytes.
       In addition, ADSCs have wide applications in tissue engineering, cell therapy and gene
       therapy.
       Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs are derived from adipose tissue at inguen of
       qualified strain C57BL/6 mice, cultured as monolayer, and cryopreserved at Sixth passage.
       These cells express specific clusters of differentiation proteins for ADSCs, and have a strong
       capacity for self-renewal while maintaining multipotency. They are tested negative for
       bacteria, fungi and mycoplasma.




Cell Characteristics and Identity
        1, Strong capacity to expand.
        2, Multipotent differentiation ability along osteogenic, chondrogenic and adipogenic lineages.
        3, Positive for CD29, CD34, CD44, and Sca-1 (> 70%), and negative for CD117 (< 5%) in
           flow cytometry assays.




Product Applications
       Adipose-derived Mesenchymal Stem Cells (ADSCs) have become a hot research target
       for their potential use in regenerative medicine and tissue engineering in areas such as
       cardiovascular, neural and orthopaedic diseases.
       Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs can be used as cell models to test and
       evaluate the immunoreactions, proliferation, immigration and differentiation of ADSCs both in
       vivo and in vitro.                                                                                3
    Cyagen OriCellTM Strain C57BL/6 Mouse Adipose-derived
    Mesenchymal Stem Cells (ADSCs) Source
           Cyagen OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells are derived
           from adipose tissue at inguen of qualified strain C57BL/6 mice and cultured in Adipose-
           derived Mesenchymal Stem Cell Growth Medium.
           Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs have been tested for:
           Exogenous Factors: Including bacterium/fungus contamination, mycoplasma contamination,
           and endotoxin contamination.
           Characteristics: Including post-thaw viability, karyotype, verification of undifferentiated state
           and differentiation potential.



    General Handling Principles
           1, Aseptic handling of the product throughout is necessary.
           2, Always freeze up several vials of Adipose-derived Mesenchymal Stem Cells (ADSCs) as
              backup once the ADSCs have been established.
           3, For all studies, using cells below passage 10 after purchasing is strongly recommended.
           4, For general maintenance of cells, if the medium becomes acidic (the pH indicator in culture
              medium appears yellow), it is recommended that the medium be changed. In general,
              change the growth medium every three days.
           5, Don’t let Strain C57BL/6 Mouse ADSCs overgrow, or it will result in contact inhibition.
              When OriCellTM Strain C57BL/6 Mouse ADSCs reach 80 to 90% confluence, it is
               recommended that the cells be subcultured.


    Preparing OriCellTM Adipose-derived Mesenchymal Stem Cell
    (ADSC) Growth Medium
           Materials Required:
           OriCellTM Adipose-derived Mesenchymal Stem Cell Growth Medium
           (Cat. No. GUXMD-90011)
           Note: Medium for strain C57BL/6 mouse ADSCs from other companies can be
           applied to the culturing of OriCellTM Strain C57BL/6 Mouse ADSCs.
           We strongly recommend that you use Cyagen tailor-made culture medium and
           other related reagents for better results.


           Kit Components:
           OriCellTM Adipose-derived Mesenchymal Stem Cell Basal Medium
           (Cat. No. GUXMD-03011-440)                                                                440 ml
           OriCellTM Adipose-derived Mesenchymal Stem Cell-qualified Fetal Bovine Serum
           (Cat. No. GUXMD-05001-50)                                                                  50 ml
           Penicillin-Streptomycin                                                                     5 ml
           Glutamine                                                                                   5 ml

           Stability/Storage:
           1, All products should be stored in the dark.
           2, OriCellTM ADSC Basal Medium is stable at 2 to 8°C for up to one year. Other components
              are stable at -20°C for up to two years. These products should be discarded beyond the
              labeled expiration date.
4          3, Once prepared, the fully supplemented complete medium can be stored for up to one
   month when stored in the dark at 2 to 8°C.
4, For optimal performance, repeated warm-cooling and freeze-thawing should be avoided.

Quality Control:
OriCellTM Adipose-derived Mesenchymal Stem Cell Growth Medium is performance tested on
OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells.
Standard evaluation includes:
1, Sterility test (bacteria, fungi and mycoplasma)
2, pH test
3, Osmolality
4, Endotoxin




                 Fig.1 OriCellTM Adipose-derived Mesenchymal Stem Cell Growth Medium




Preparation of Complete Medium
1, Prior to use, thaw OriCellTM Mouse Adipose-derived Stem Cell-qualified Fetal Bovine Serum
   under refrigeration (2 to 8°C) over night or until completely thawed. Gently swirl the
   bottle to ensure homogeneity. OriCellTM Adipose-derived Stem Cell-qualified Fetal Bovine
   Serum has been heat-inactivated and is ready to use after thawing.
Note: The thawed serum may contain some flocculent precipitates. The presence
     of these substances in serum does not alter the performance characteristics of
     the product. It is not recommended to filter the serum to remove these
     precipitates. Doing so may result in the loss of some serum nutrients.
2,   About 30 minutes prior to use, thaw Penicillin-Streptomycin solution and Glutamine
     solution at room temperature. Gently swirl the vials to ensure homogeneity.
3,   Disinfect with 70% v/v ethanol the external surfaces of the bottles/vials for every
     component in the kit. Allow ethanol to evaporate away.
4,   In a laminar flow hood aseptically open the bottles/vials.
5,   Transfer the entire amount of OriCellTM Adipose-derived Mesenchymal Stem Cell-qualified
     Fetal Bovine Serum, Penicillin-Streptomycin solution and Glutamine solution into OriCellTM
     ADSC Basal Medium.
6,   Rinse each vial with the medium and transfer the rinse medium back to the bottle of basal
     medium.
7,   Gently swirl the fully supplemented complete medium to ensure a homogeneous mixture.
     The complete medium is now ready to use.
Note: Although each component in this kit is supplied sterile, it is strongly
     recommended that the fully supplemented complete medium be filtered.                         5
    Thawing and Establishing OriCellTM Strain C57BL/6 Mouse
    Adipose-derived Mesenchymal Stem Cells (ADSCs) Using Cyagen
    OriCellTM ADSC Growth Medium
           Materials Required:
           Trypsin-EDTA (Cat. No. TEDTA-10001-100)
           Phosphate-Buffered Saline (1×PBS) (Cat. No. PBS-10001-500)
           OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells
           (Cat. No. MUBMD-01001)
           OriCellTM Adipose-derived Mesenchymal Stem Cell Growth Medium
           (Cat. No. GUXMD-90011)
           Note: Medium for strain C57BL/6 mouse ADSCs from other companies can be
           applied to the culturing of OriCellTM Strain C57BL/6 Mouse ADSCs.

           We strongly recommend that you use Cyagen tailor-made culture medium and other related
           reagents for better results.
           1, Prepare 37°C water bath and pre-warm OriCellTM ADSC Growth Medium to 37°C.
           2, Add 9 ml of OriCellTM ADSC Growth Medium to a 15 ml conical tube.
           3, Remove the cryovial of OriCellTM Strain C57BL/6 Mouse ADSCs from liquid nitrogen. Quickly
              thaw the vial in 37°C water bath until the last crystal piece disappears, and finish the
              thawing procedure within 3 minutes. Be careful not to submerge the entire vial. Maximum
              cell viability is dependent on the rapid and complete thawing of frozen cells.
           Note: Thawing the cells for longer than 3 minutes results in less than optimal
              results.
           4, As soon as the cells are completely thawed, disinfect the outside of the vial with 70% v/v
              ethanol.
           5, In a laminar flow hood, use pipette to transfer the cells to the conical tube containing
              OriCellTM ADSC Growth Medium. Be careful not to introduce any bubbles during the
              transfer process.
           6, Rinse the vial with 1 ml of medium to reduce the loss of cell and then transfer this 1 ml
              of cell suspension to the conical tube.
           7, Gently mix the cell suspension by slowly pipeting up and down. Be careful not to introduce
              any bubbles.
           8, Centrifuge the cell suspension at 250g for 5 minutes.
           9, Carefully aspirate as much of the supernatant as possible and add 2-3 ml of fresh OriCellTM
              ADSC Growth Medium (pre-warmed to 37°C).
           10, Gently re-suspend the cells in OriCellTM ADSC Growth Medium.
           11, Plate the cells into a T25 flask and add sufficient OriCellTM ADSC Growth Medium. Gently
                rock the culture flask to evenly distribute the cells.
           12, Incubate at 37°C in a 5% CO2 humidified incubator.
           13, The next day, change the medium with fresh OriCellTM ADSC Growth Medium (pre-warmed
                to 37°C).
           14, Change the growth medium every three days thereafter.
           15, When the cells are approximately 80 to 90% confluence, they can be dissociated with
                Trypsin-EDTA and passaged.

           Note: Changing Medium
           1, Warm an appropriate amount of medium to 37°C in a sterile container. Remove the
              medium and replace it with the warmed, fresh medium and return the flask to the
              incubator.
           2, Avoid repeated warming and cooling of the medium. If the entire contents are not needed
6             for a single procedure, transfer only the required volume to a sterile secondary container.
                     Figure 2. OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal
                                          Stem Cells are established




Passaging Cyagen OriCellTM Strain C57BL/6 Mouse Adipose-
derived Mesenchymal Stem Cells (ADSCs)

       Materials Required:
       Trypsin-EDTA (Cat. No. TEDTA-10001-100)
       Phosphate-Buffered Saline (1× PBS) (Cat. No. PBS-10001-500)
       OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells
       (Cat. No. MUBMD-01001)
       OriCellTM Adipose-derived Mesenchymal Stem Cell Growth Medium
       (Cat. No. GUXMD-90011)
       Note: Medium for strain C57BL/6 mouse ADSCs from other companies can be
       applied to the culturing of OriCellTM Strain C57BL/6 Mouse ADSCs.

       We strongly recommend that you use Cyagen tailor-made culture medium and other related
       reagents for better results.
       1, Prewarm the OriCellTM ADSC Growth Medium, 1×PBS, and Trypsin-EDTA solution to 37°C.
       2, Carefully aspirate spent medium from the 80 to 90% confluent monolayer of OriCellTM
          Strain C57BL/6 Mouse ADSCs.
       3, Add 1×PBS (6 ml for T25 flask, 3 ml for T25 flask). Be careful not to disturb the
          monolayer. Rinse the monolayer by gently rocking the flask back and forth.
       4, Aspirate 1× PBS and discard.
       5, Repeat the step 3-4 two or three times.
       6, Add Trypsin-EDTA solution (1.5 ml for T75 flask, 0.5 ml for T25 flask). Gently rock the
          flask back and forth to ensure that the entire monolayer is covered with the Trypsin-EDTA
          solution. Allow the trypsinization to continue until the majority of the cells (approximately
          80%) are rounded up. At this point, gently tap the side of the flask to release the majority
          of cells from the culture surface.
       Note: Avoid leaving cells exposed to the trypsin longer than necessary. Care should
          also be taken that the cells not be forced to detach prematurely, as this may
          result in clumping.
       7, After the cells are visibly detached, immediately add OriCellTM ADSC Growth Medium (pre-
          warmed to 37°C) (6 ml for T75 flask, 3 ml for T25 flask) to neutralize the trypsinization.      7
    8, Gently pipet the medium over the cells to dislodge and re-suspend the cells. Repeat 5-6
       times until all the cells are dissociated from the flask and evenly dispersed into a single
       cell suspension.
    9, Transfer the dissociated cells into a 15 ml conical tube.
    10, Centrifuge at 250g for 5 minutes to pellet the cells.
    11, Carefully aspirate as much of the supernatant as possible.
    12, Add 2 ml of OriCellTM ADSC Growth Medium to the conical tube and re-suspend the cells
         thoroughly but gently.
    13, Plate the cells into appropriate flasks. OriCellTM Strain C57BL/6 Mouse ADSCs can be split
         at 1:2 or other appropriate ratio.
    14, Add sufficient medium.
    15, Incubate the cells at 37°C in a 5% CO2 humidified incubator.
    Note: Care should be taken to avoid introducing bubble during pipeting.

    Hints
    Time to Change Medium
    Although the cells do not reach 80 to 90% confluence, if the medium becomes acidic (the
    pH indicator in culture medium appears yellow), it is recommended that the medium be
    changed. In general, change the growth medium every three days.
    Time to Subculture
    When OriCellTM Strain C57BL/6 Mouse ADSCs reach 80 to 90% confluence, it is recommended
    that the cells be subcultured. Don’t let OriCellTM Strain C57BL/6 Mouse ADSCs overgrow, or it
    will result in contact inhibition.




            Passage 3-40x                    Passage 5-40x                    Passage 7-40x




            Passage 3-100x                  Passage 5-100x                    Passage 7-100x

             Figure 3. Images of OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal
                                    Stem Cells at passage 3, 5 and 7




8
OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal
Stem Cells (ADSCs) Differentiation with Cyagen OriCellTM
Differentiation Medium
       Cyagen OriCellTM ADSCs can differentiate into a variety of cell types, including osteocytes,
       adipocytes, and chondrocytes.


       Osteogenic Differentiation

       Materials Required:
       OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium
       (Cat. No. GUXMX-90021)

       Kit Components:
       OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Basal Medium
       (Cat. No. GUXMX-03021-175)                                                               175 ml
       OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum
       (Cat. No. GUXMX-05001-20)                                                                 20 ml
       Penicillin-Streptomycin                                                                     2 ml
       Glutamine                                                                                   2 ml
       Ascorbate                                                                                 400μl
       β-Glycerophosphate                                                                         2 ml
       Dexamethasone                                                                               20μl
       Alizarin Red S                                                                             10 ml




                     Fig. 4 OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium




       Stability/Storage:
       1, All products should be stored in the dark.
       2, OriCellTM Osteogenic Differentiation Basal Medium is stable at 2 to 8°C for up to one year.
          Other components are stable at -20°C for up to two years. hese products should be
          discarded beyond the labeled expiration date.
       3, Once prepared, the fully supplemented complete medium can be stored for up to one
          month when stored in the dark at 2 to 8°C.
       4, For optimal performance, repeated warm-cooling and freeze-thawing should be avoided.            9
     Quality Control
     OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium is performance tested on
     OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells.
     Standard evaluation includes:
     1, Sterility test (bacteria, fungi and mycoplasma)
     2, pH test
     3, Osmolality
     4, Endotoxin

     Preparation of Complete Medium
     1, Prior to use, thaw OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum under
        refrigeration (2 to 8°C) over night or until completely thawed. Gently swirl the bottle to
        ensure homogeneity. OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum has
        been heat-inactivated and is ready to use after thawing.
     Note: The thawed serum may contain some flocculent precipitates. The presence
        of these substances in serum does not alter the performance characteristics of
        the product. It is not recommended to filter the serum to remove these
        precipitates. Doing so may result in the loss of some serum nutrients.
     2, About 30 minutes prior to use, thaw Ascorbate, β-Glycerophosphate, Penicillin-
        Streptomycin solution and Glutamine solution at room temperature. Gently invert the vials
        several times to ensure homogeneity.
     Note: Centrifuge the vials briefly at low speed (5,000g) before removing the caps
        to ensure recovery of entire content.
     3, About 10 minutes prior to use, thaw Dexamethasone at room temperature.
     Note: Centrifuge the vial briefly at low speed (5,000g) before removing the cap to
        ensure recovery of entire content.
     4, Disinfect with 70% v/v ethanol the external surfaces of the bottles/vials for every
        component in the kit. Allow ethanol to evaporate away.
     5, In a laminar flow hood aseptically open the bottles/vials.
     6, Transfer the entire amount of Ascorbate, β-Glycerophosphate, OriCellTM Mesenchymal Stem
        Cell-qualified Fetal Bovine Serum, Penicillin-Streptomycin solution and Glutamine solution
        into OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Basal Medium.
     7, Rinse each vial/bottle with the medium and transfer the rinse medium back to the bottle
        of basal medium.
     8, To transfer the entire amount of Dexamethasone, add 0.5 ml medium to the vial, mix by
        pipeting and then transfer the mixture back to the bottle of basal medium as much as
        possible.
     9, Repeat step 8 several times.
     10, Gently swirl the fully supplemented complete medium to ensure a homogeneous mixture.
          The complete medium is now ready to use.
     Note: Although each component in this kit is supplied sterile, it is strongly
        recommended that the fully supplemented complete be filtered.

     Osteogenesis Protocol (for 6-well tissue culture plate):
     1, OriCellTM Starin C57BL/6 Mouse ADSCs are cultured in OriCellTM ADSC Growth Medium
        (growth medium thereafter) at 37°C in a 5% CO2 humidified incubator.
     2, When cells are approximately 80-90% confluent, they can be dissociated with Trypsin-
        EDTA.
     3, OriCellTM Starin C57BL/6 Mouse ADSCs are replated in OriCellTM ADSC Growth Medium at
        3 x 103 cells/cm2 in 6-well tissue culture plates with a medium volume of 2 ml per well.
     4, Incubate the cells at 37°C in a 5% CO2 humidified incubator.
     5, After 24 hours, carefully aspirate off the growth medium from each well and add 2 ml
        Mesenchymal Stem Cell Osteogenic Differentiation Medium.
10   6, Refeed cells every 3 days for 2-3 weeks by completely replace the medium with fresh
   Mesenchymal Stem Cell Osteogenic Differentiation Medium.
7, After 2-3 week differentiation, cells can be fixed and stained with Alizarin Red S.


Alizarin Red Stain Analysis
1, After differentiating, remove Osteogenic Differentiation Medium from well and rinse with
   1×PBS.Fix cells with 2ml of 4% formaldehyde solution for 30 minutes.
2, Then rinse wells twice with 1×PBS and stain cells with 1ml Alizarin Red working solution
   for 3 to 5 minutes.
3, Rinse wells 2-3 times with 1×PBS, then visualize under light microscope and capture
   images.




         Fig. 5 OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells are
                   differentiated to Osteocytes and are stained with Alizarin Red S.




Adipogenic Differentiation
Materials Required:
OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Medium
(Cat. No. GUXMX-90031)

Kit Components:
OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Basal Medium A:
(Cat. No. GUXMX-03031-200)                                                                  200 ml
OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum
(Cat. No. GUXMX-05001-20)                                                                   20 ml
Penicillin-Streptomycin                                                                       2 ml
Glutamine                                                                                     2 ml
Insulin                                                                                     200 μl
IBMX                                                                                        200 μl
Indomethacin                                                                                200 μl
Dexamethasone                                                                               200 μl
OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Medium B:
(Cat. No. GUXMX-03032-200)                                                                  200 ml
OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum
(Cat. No. GUXMX-05001-20)                                                                   20 ml
Penicillin-Streptomycin                                                                      2 ml
Glutamine                                                                                    2 ml
Insulin                                                                                     200 μl
Oil Red O                                                                                   10 ml    11
                  Fig. 6 OriCellTM Mesenchymal Stem Cells Adipogenic Differentiation Medium




     Stability/Storage:
     1, All products should be stored in the dark.
     2, OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Basal Medium A&B is stable at
        2 to 8°C for up to one year. Other components are stable at -20°C for up to two years.
        These products should be discarded beyond the labeled expiration date.
     3, Once prepared, the fully supplemented complete medium can be stored for up to one
        month when stored in the dark at 2 to 8°C.
     4, For optimal performance, repeated warm-cooling and freeze-thawing should be avoided.

     Quality Control:
     OriCellTM Mesenchymal Stem Cell Differentiation Medium is performance tested on OriCellTM
     Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells.
     Standard evaluation includes:
     1, Sterility test (bacteria, fungi and mycoplasma)
     2, pH test
     3, Osmolality
     4, Endotoxin



     Preparation of Mesenchymal Stem Cell Adipogenic Differentiation Medium
     A (induction medium)
     1, Prior to use, thaw OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum at 2 to 8°C
        under refrigeration until thawed. Gently swirl the bottle to ensure homogeneity. OriCellTM
        Mesenchymal Stem Cell-qualified Fetal Bovine Serum has been heat-inactivated and is
        ready to use after thawing.
     Note: The thawed serum may contain some flocculent precipitates. The presence
       of these substances in serum does not alter the performance characteristics of
       the product. It is not recommended to filter the serum to remove these
        precipitates. Doing so may result in the loss of some serum nutrients.
     2, About 30 minutes prior to use, thaw Dexamethasone, Insulin, IBMX, Indomethacin,
        Penicillin-Streptomycin solution and Glutamine solution at room temperature. Gently invert
        the vials to ensure homogeneity.
     Note: Centrifuge the vials briefly at low speed before removing the caps to ensure
12     recovery of entire content.
3, Disinfect with 70% v/v ethanol the external surfaces of the bottles/vials for every
   component in the kit. Allow ethanol to evaporate away.
4, In a laminar flow hood aseptically open the bottles/vials.
5, Transfer the entire amount of OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine
   Serum, Penicillin-Streptomycin solution and Glutamine solution into OriCellTM Mesenchymal
   Stem Cell Adipogenic Differentiation Basal Medium A.
6, Rinse each vial with the medium and transfer the rinse medium back to the bottle of basal
   medium A.
7, Transfer the entire amount of Dexamethasone, Insulin, IBMX and Indomethacin into
   OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Basal Medium A.
8, Rinse each vial with the medium and transfer the rinse medium back to the bottle of basal
   medium A as much as possible.
9, Repeat step 8 several times.
10, Gently swirl the fully supplemented complete medium to ensure a homogeneous mixture.
     The complete medium is now ready to use.
Note: Although each component in this kit is supplied sterile, it is strongly
   recommended that the fully supplemented complete medium be filtered.




Preparation of OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation
Medium B (maintenance medium)
1, Prior to use, thaw OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine Serum at 2 to 8°C
   under refrigeration until thawed. Gently swir the bottle to ensure homogeneity. OriCellTM
   Mesenchymal Stem Cell-qualified Fetal Bovine Serum has been heat-inactivated and is
   ready to use after thawing.
Note: The thawed serum may contain some flocculent precipitates. The presence
   of these substances in serum does not alter the performance characteristics of
   the product. It is not recommended to filter the serum to remove these
   precipitates. Doing so may result in the loss of some serum nutrients.
2, About 30 minutes prior to use, thaw Insulin, Penicillin-Streptomycin solution and
   Glutamine solution at room temperature. Gently invert the vials to ensure homogeneity.
Note: Centrifuge the vials briefly at low speed before removing the caps to ensure
   recovery of entire content.
3, Disinfect with 70% v/v ethanol the external surfaces of the bottles/vials for every
   component in the kit. Allow ethanol to evaporate away.
4, In a laminar flow hood aseptically open the bottles/vials.
5, Transfer the entire amount of OriCellTM Mesenchymal Stem Cell-qualified Fetal Bovine
   Serum, Penicillin-Streptomycin solution and Glutamine solution into OriCellTM Mesenchymal
   Stem Cells Adipogenic Differentiation Basal Medium B.
6, Rinse each vial with the medium and transfer the rinse medium back to the bottle of basal
   medium B.
7, Transfer the entire amount of Insulin into OriCellTM Mesenchymal Stem Cell Adipogenic
   Differentiation Basal Medium B.
8, Rinse the vial with the medium and transfer the rinse medium back to the bottle of basal
   medium B as much as possible.
9, Repeat step 8 several times.
10, Gently swirl the fully supplemented complete medium to ensure a homogeneous mixture.
     The complete medium is now ready to use.
Note: Although each component in this kit is supplied sterile, it is strongly
  recommended that the fully supplemented complete medium be filtered.                           13
     Adipogenesis Protocol (for 6-well tissue culture plate):
     1, OriCellTM Strain C57BL/6 Mouse ADSCs are cultured in OriCellTM ADSC Growth Medium
        (growth medium thereafter) at 37°C in a 5% CO2 humidified incubator.
     2, When cells are approximately 80-90% confluent, they can be dissociated with Trypsin-
        EDTA.
     3, OriCellTM Strain C57BL/6 Mouse ADSCs are replated in growth medium at 2x104 cells/cm2
        in 6-well tissue culture plates with a medium volume of 2 ml per well.
     4, Incubate the cells at 37°C in a 5% CO2 humidified incubator.
     5, Refeed the cells every 3 days until they are 100% confluent or post confluent. It’s strongly
        suggested to induce the adipogenic differentiation of OriCellTM Strain C57BL/6 Mouse
        ADSCs 3-5 day post confluent.
     6, When the cells reach 100% confluent or post confluent, carefully aspirate off the spent
        growth medium from the wells and add 2 ml induction medium per well.
     7, Three days later, change the medium to maintenance medium by completely replacing the
        spent induction medium.
     8, 24 hours later, change the back to induction medium.
     9, To optimally differentiate OriCellTM Strain C57BL/6 Mouse ADSCs into adipogenic cells,
        repeat the cycle of induction/maintenance three times.
     10, After the three cycle of induction/maintenance, culture the cells in maintenance medium
          for additional 7 days by replacing the medium every 3 days.


     Oil Red O Stain Analysis
     1, After differentiating, remove Adipogenic Differentiation Medium from well and rinse with
       1×PBS. Fix cells with 2ml of 4% formaldehyde solution for 30 minutes.
     2, Then rinse wells twice with 1×PBS and stain cells with 1ml Oil Red O working solution (3:2
        dilution with distilled water and Filter with filter paper) for 30 minutes.
     3, Rinse wells 2-3 times with 1×PBS, then visualize under light microscope and capture
        images.




              Fig.7 OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells are
                         differentiated to adipocytes and are stained with Oil Red O.




14
Chondrogenic Differentiation

Materials Required:
OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium
(Cat. No. GUXMX-90041)

Kit Component:
OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Basal Medium
(Cat. No. GUXMX-03041-194)                                                                 97 ml
Dexamethasone                                                                               10μl
Ascorbate                                                                                  300μl
ITS + Supplement                                                                            1 ml
Sodium Pyruvate                                                                            100μl
Proline                                                                                    100μl
TGF-β3                                                                                      1 ml
Alcian Blue                                                                                10 ml




              Fig. 8 OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium




Stability/Storage:
1, OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Basal Medium is stable at 2
   to 8°C for up to one year. ITS + Supplement is stable for a minimum three months at 2 to
   8°C, Other components are stable at -20°C for up to two years. These products should be
   discarded beyond the labeled expiration date.
2, Once prepared, the fully supplemented complete medium can be stored for 12 hours when
   stored in the dark at 2 to 8°C.
3, For optimal performance, repeated warm-cooling and freeze-thawing should be avoided.

Quality control:
OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium is performance tested
on C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells.
Standard evaluation includes:
1, Sterility test (bacteria, fungi and mycoplasma)
2, pH test
3, Endotoxin                                                                                       15
     Prepare Incomplete Chondrogenic Induction Medium
     1, Decontaminate the external surface of High glucose DMEM and the following components
        with 70% v/v ethanol.
        a. Dexamethasone
        b. Ascorbate
        c. ITS + Supplement
        d. Sodium Pyruvate
        e. Proline
     2, Aseptically open the above components and add the contents to OriCellTM Mesenchymal
        Stem Cell Chondrogenic Differentiation Basal Medium to prepare the Incomplete
        Chondrogenic Induction Medium.
     3, Rinse each vial with the medium. It may not be possible to recover the entire content of
        each component. Small losses should not affect cell characteristics.
     4, Store the Incomplete Chondrogenic Induction Medium at 2 to 8°C in the dark until needed.
     Aliquot TGF-β3:
     Aliquot small volumes of TGF-β3 into freezer-safe tubes and store at less than
     –70°C for no more than 6 months.
     Note: 10 μl of TGF-β3 will convert 1 ml of Incomplete Chondrogenic Medium into
     Complete Medium.

     Prepare Complete Chondrogenic Induction Medium:
     1, After thawing, the aliquot of TGF-β3 may need to be centrifuged briefly at low speed to
        pull the small volume to the bottom of the tube.
     2, Pipette the volume of Incomplete Chondrogenic Induction Medium that you intend to
        supplement (e.g. 10 ml) into a tube.
     3, To recover the full volume of TGF-β3, transfer 100 μl of this Incomplete Chondrogenic
        Medium to the tube of TGF-β3.
     4, Mix the solution by pipeting and transfer it back to the tube of Chondrogenic Induction
        Medium. Repeat this process to be certain that you have recovered the TGF-β3. Cap and
        invert several times to mix.
     5, The Chondrogenic Induction Medium is now complete.
     Note: Complete Chondrogenic Medium must be prepared fresh and used within 12
       hours.

     Chondrogenesis Protocol:
     1, Calculate the total number of pellet cultures required for your experiment (2.5×105 ADSCs
        are needed to form each chondrogenic pellet). Transfer this amount of cells to an
        appropriate culture tube to wash the cells.
     2, Wash the OriCellTM Strain C57BL/6 Mouse ADSCs with Incomplete Chondrogenic Medium:
        Centrifuge the cells at 150g for 5 minutes at room temperature, and aspirate/discard
        the supernatant. Resuspend the cells in 1 ml Incomplete Chondrogenic Medium per 7.5×105
        cells, centrifuge again at 150g for 5 minutes and aspirate/discard the medium.
     3, Resuspend the ADSCs in Complete Chondrogenic Medium to a concentration of 5.0×105
        cells per ml.
     4, Aliquot 0.5 ml (2.5×105 cells) of the cell suspension into 15 ml polypropylene culture
        tubes. Centrifuge the cells at 150g for 5 minutes at room temperature.
     Note: DO NOT aspirate the supernatant or resuspend the pellet.
     5, Loosen the caps of the tubes one half turn to allow gas exchange and incubate the tubes
        at 37°C, in a humidified atmosphere of 5% CO2. Do not disturb the pellets for 24 hours.
     6, Feed the cell pellets every 2-3 days by completely replacing the medium in each tube (to
        avoid aspirating the pellets when aspirating the medium, attach a sterile 1-200 μl
        pipette tip to the end of the aspirating pipette). Add 0.5 ml of freshly prepared Complete
16      Chondrogenic Medium to each tube.
       7, After replacing the medium, flick the bottom of each tube to ensure that the pellet is free-
          floating, loosen the caps and return the tubes to the 37°C incubator.
       8, Chondrogenic pellets should be harvested after 14 to 28 days in culture. Pellets may be
          formalin fixed and paraffin embedded for Alcian Blue stain.


       Alcian Blue Staining Procedure
       1, Fixation: formalin fixed, paraffin embedded tissue sections.
       2, Staining procedure:
          a) Deparaffinize slides and hydrate to distilled water.
          b) Stain in Alcian Blue solution for 30 minutes.
          c) Wash in running tap water for 2 minutes.
          d) Rinse in distilled water.
          e) Visualize under light microscope, and capture images for analysis. Blue staining
             indicates synthesis of proteoglycans by chondrocytes.




                  Fig.7 OriCellTM Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells are
                           differentiated to chondrocytes and are stained with Alcian Blue.




Cryopreservation of ADSCs using OriCellTM NCR Protein-Free
Cryopreservation Medium
       OriCellTM NCR Protein-free Cryopreservation Medium is a protein-free freezing medium for
       eukaryotic cells. It is formulated by defined chemical composition and can be used directly.
       The cells can be frozen directly in -80°C without programmable freezer or stepped cooling
       down.

       Features
       1,   Special for Mesenchymal Stem Cells.
       2,   High cell viability.
       3,   Protein-free. Defined chemical composition.
       4,   Batch to batch stability.
       5,   Direct freezing at -80°C. No need for programmable freezer or stepped cooling down.


       Packing
       50ml/ Bottle
       20ml/ Bottle                                                                                      17
     Stability/Storage:
     Store below 4°C. The product is stable below 4°C for up to 3 years. These products should be
     discarded beyond the labeled expiration date.
     For optimal performance, repeated warm-cooling and freeze-thawing should be avoided.




                         Fig.10 OriCellTM NCR Protein-Free Cryopreservation Medium.




     Quality Control:
     OriCellTM NCR Protein-free Cryopreservation Medium is performance tested on OriCellTM Strain
     C57BL/6 Mouse ADSCs.
     Standard evaluation includes:
     1, Sterility test (bacteria, fungi and mycoplasma)
     2, pH test
     3, Osmolality
     4, Endotoxin
     5, Pyrogen

     Materials Required:
     NCR Protein-Free Cryopreservation Medium (Cat. No. NCPF-10001)

     Note: Culture logarithmic growth phase stem cells, replace with fresh medium 24 h
        before freezing.
     1, Select the cells in logarithmic growth phase according to the methods used to collect cells
        in vitro. Perform a cell count to determine the viable cell density.
     2, Centrifuge the cells 3~5minutes at 250g, 20°C. Remove the supernatant using a pipette.
     3, Resuspend the precipitate with Cryopreservation Medium at the cell density of 1ml for
        105~106 cells.
     4, Dispense aliquots of the cell suspension into cryogenic storage vials that are properly
        labeled.
     5, Place the vials directly in a -80°C refrigerator and transfer the frozen vials to liquid
        nitrogen for preservation after 24 hours.




18
Appendix

Trouble Shooting
The table below lists some potential problems and solutions that help you troubleshoot your
OriCellTM Strain C57BL/6 Mouse ADSCs cultures.

     Problem                    Cause
                     The storage             not        Purchase a replacement, and store in
                        meet the requirements           liquid nitrogen for long-                    .
                      Thawing the cells takes too       Control the thawing procedure for
                                                        no more than 3 minutes.
                                                                           wash the tube with
 Low cell recovery      Cells are incompletely
                                                        culture medium twice and transfer
       rate                    aspirated
                                                        all cells to the dish.
                                                        Care should be taken to avoid introducing
                      Cells are handled roughly         bubbles during         ; avoid vortex or
                                                        high-speed centrifuge.
                     Medium is not pre-warmed           Warm medium to 37°C before recovery.
                                                        Discard the cells         , and disinfect
                                                    .   the experimental environment before
                                                        recovering a new stock.
                                                        Wash the cells with PBS for 2-
  Slow cell growth                                      remove serum (serum will inhibit the
                            Over di
                                                                   trypsin).
                                                        Control the                .



                                                        Use Cyagen tailor-made culture medium.
                       Inappropriate serum and
                                                        If other serum and medium products are
                              medium
                                                        used, please do pre-screening test.

                     Dead cells are not removed
                                                        recovery to ensure removal of all dead
                              promptly
                                                        cells.
                                                        Discard the cells         , and disinfect
     Cell aging                                         the experimental environment before
                                                        recovering.
                                                        Some stem cells can secrete factors to
                                                        support the cell growth. Therefore, a
                                                                                                be
                                                        maintained, otherwise it will lead to cell
                                                                                             cell
                                                        aging.


                                                                                                         19
                                                         Wash the cells with PBS for 2-
                                                         Remove serum (serum will inhibit the
                                  Over
                                                                    trypsin).
          Cell aging                                     Control the                 .
                                                         The cells should be subcultured when
                                                  not
                                                         reaching                          there
                                  appropriate
                                                         will be                 .
         Cells show
                            DMSO is not completely       Wash the cells with pre-warmed medium
        spontaneous
                          removed during cell recovery   for 2-       during recovery.

         I                                               Use Cyagen tailor-made
                of Cell                                  medium.
                             Cell passage is too high    Use cells at low passage.




     Related products
     1, Trypsin-EDTA
        (Cat. No. TEDTA-10001-100)
     2, Phosphate-Buffered Saline (1 × PBS)
        (Cat. No. PBS-10001-500)
     3, OriCellTM Strain C57BL/6 mouse Adipose-derived Mesenchymal Stem Cells
        (Cat. No. MUBMD-01001)
     4, OriCellTM Adipose-derived Mesenchymal Stem Cell Growth Medium
        (Cat. No. GUXMD-90011)
     5, OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium
        (Cat. No. GUXMX-90021)
     6, OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Medium
        (Cat. No. GUXMX-90031)
     7, OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium
       (Cat. No. GUXMX-90041)
     8, OriCellTM NCR Protein-Free Cryopreservation Medium
        (Cat. No. NCPF-10001)


     Purchaser's Notification
     For research use only. Not intended for any animal or human therapeutic or diagnostic use.




20

								
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