Protocol:

Isolation, Expansion,

Multilineage Differentiation,

and Identification of MSC

Isolation of MSCs

Isolation and culture of adipose MSCs

Isolation and culture of bone marrow MSCs

Isolation and culture of human umbilical MSCs

Expansion of MSCs

MSC Cell Recovery

MSC cell subculture

MSC cell cryopreservation

Multilineage Differentiation of MSCs

Osteogenic Differentiation of MSCs

Adipogenic Differentiation of MSCs

Chondrogenic Differentiation of MSCs

Identification Markers of MSCs

Surface markers of MSCs

Identification by flow cytometry

MSCs Culture Cytokines Isolation, Expansion, Multilineage Differentiation, and Identification of MSC

Contents

P/ 01

P/ 04

P/ 05

P/ 07

P/ 09

Collect the

adipose

tissue

Wash with

PBS

Shake and

digest with

digestive

enzymes

Centrifugal

filtration Seed

< Enzymatic digestion method for the isolation of adipose MSCs >

Isolation of MSCs

Isolation and culture of adipose MSCs

Mouse adipose tissue harvesting

1. Euthanize mice aged 3-28 days (young pups are recommended) by cervical dislocation. Soak the mice in 75%

alcohol for 10 minutes.

2. Place the mouse horizontally with its back facing upwards. Make a 1 cm vertical incision in the center of the

back. Tear off the groin skin along the incision to expose the symmetrical white adipose tissue in the groin area,

and perform blunt dissection.

Isolation and culture of adipose MSCs (Enzymatic digestion method)

1. Digestion: Collect 25 mL of adipose tissue; Rinse the tissue 3 times with 4℃ PBS; Remove the fascia and red

blood cells; Cut the tissue into pieces with ophthalmic scissors; Rinse 3 times with 4℃ PBS; Transfer 5 mL of

adipose tissue into each of 50 mL centrifuge tubes; Add twice the volume of 0.075% type I collagenase; Shake

and digest in a 37℃ constant temperature shaker at a speed of 80 r/min for 1 hour. After thorough digestion, add

an equal volume of DMEM-F12 complete medium containing 10% FBS to terminate the digestion.

2. Centrifugation and Counting: Centrifuge at 300 g for 10 minutes; Remove the undigested adipose tissue from

the upper layer, grease and the supernatant from the middle layer; Resuspend the precipitate in 5 mL of red

blood cell lysis buffer; Let stand at room temperature for 5 minutes; Centrifuge at 200 g for 5 minutes; Remove

the supernatant and resuspend the precipitate in complete medium; Filter through a 200-mesh sieve, collect

the sample and perform cell counting; Adjust the cell concentration according to the counting result, then seed

in a culture dish at a density of (1-5)×104/cm2; Culture in an incubator at 37℃ with 5% CO2.

3. Seeding and Cultivation: Change the medium for the first time after 48 hours then change the medium every

3 days; When the cells reach 80%-90% confluence, digest with 0.25% trypsin and pass the cells at a ratio of 1:2.

Isolation, Expansion, Multilineage Differentiation,

and Identification of MSC

Mesenchymal Stem Cells（MSCs）are a type of pluripotent stem cell derived from the mesoderm. They can be

isolated from various tissues, including bone marrow, adipocyte, umbilical cord, placenta, and skin. MSCs exhibit

strong proliferation ability and multi-differentiation potential. They can differentiate into osteoblasts, chondrocytes,

adipocytes, as well as muscle cells, liver cells, pancreatic islet cells and other cell types.

MSCs have shown great application prospects in tissue engineering, gene therapy and immunotherapy. They have

brought new hopes and breakthrough directions to the medical field, and hold significance potential for the treatment and research on various diseases.

SVF

SVF: Stromal vascular fraction, mainly contains adipose mesenchymal stem cells, hematopoietic stem cells,

endothelial progenitor cells, etc.

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Isolation and culture of mouse bone marrow MSCs (whole-bone-marrow method, bone fragment method)

1. Collect 4 to 6 mice aged 2 to 3 weeks; Euthanize the mice by cervical dislocation; Soak the mice in 75% ethanol

for 5 minutes; Cut open the skin in the groin area; Pull and strip the epidermis downward along the opening;

Remove the bilateral femurs and tibias under sterile conditions; Remove the muscles and connective tissues

around the femurs and tibias; Place the bones in a sterile culture dish containing PBS and rinse 3 times.

2. Cut off both ends of the femurs and tibias to expose the bone marrow cavity; Use a 10 mL syringe to draw the

bone marrow MSC culture medium and rinse the bone marrow cavity repeatedly until the bone turns white.

3. Whole-bone-marrow method: Collect the bone marrow suspension and place in a centrifuge tube; Centrifuge at 200 g, 4℃ for 5 minutes; Resuspend with culture medium; Adjust the cell concentration to 1×106 cells/mL

and seed in T25 culture flasks; Place the flasks in a 37℃, 5% CO2 incubator for culture; Change the medium every

24 hours for 3 days; Then, change the medium every 2-4 days according to the cell growth rate; When the

confluence reaches 80%-90%, passage at a ratio of 1:2.

4. Bone Fragment Method: Cut the femurs and tibias from which the bone marrow has been washed out into 1-3

mm3 fragments; Digest the bone fragments with complete medium (containing 5% FBS and 1.0 mg/ml type II

collagenase) in a 37℃ shaker at 200 rpm for 1-2 hours. Stop digestion when the bone fragments become loose;

Discard the supernatant digestive fluid; Rinse the bone fragments three times with the complete medium; Seed

the bone fragments in the culture dish and add an appropriate amount of fresh complete medium (containing

10% FBS) to submerge the bone fragments; Place the culture dish in an incubator at 37℃ and 5% CO2. Change

the culture medium every 2-3 days; When the confluence reach 80%-90%, digest with trypsin-EDTA solution

(0.25%:0.02%) and passage at a ratio of 1:3.

Isolation and culture of bone marrow MSCs

1. Extract 5mL of bone marrow fluid under sterile conditions; Place

the extracted bone marrow in a blood collection tube containing

anticoagulant; Conduct the experiment within 24 hours after

collection; Dilute and mix the extraction by adding an equal amount

of PBS; Centrifuge at 300 g for 10 minutes; Discard the adipose layer;

Slowly add the diluted bone marrow fluid along the wall of the

centrifuge tube on top of the Ficoll separation solution (1.077 g/mL)

of the same volume; Centrifuge at 800 g for 30 minutes.

2. Carefully aspirate the bone marrow-derived mononuclear cells

(BMMNCs); Add twice the volume of PBS for suspension; Centrifuge

at 300 g for 10 minutes; Discard the supernatant and repeat the

washing step twice.

3. Resuspend the cells with the complete medium of BMSCs. Seed

the cells in T25 cell culture flasks at a density of 2×105/mL; Place the

flasks in an incubator at 37℃ and 5% CO2; Change the medium for

the first time after 3 days to remove the suspended cells, and then

change the medium once every 2-3 days; Monitor the morphology

and growth of the cells; When the confluence reaches 80%-90%

(about 12 days), digest with trypsin-EDTA solution (0.25%: 0.02%)

and passage at a ratio of 1:2.

doi:10.1038/nprot.2009.238

doi:10.1007/978-1-4939-3584-0_1

Before centrifugation

Ficoll separation solution

Dilution of bone marrow fluid

Plasma

BMMNC

Ficoll separation solution

Erythrocytes,etc

After centrifugation

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Isolation and culture of human bone marrow MSCs (density gradient centrifugation method)

Density gradient centrifugation method for the

isolationof bone marrow MSCs

Isolation and culture of human umbilical MSCs (tissue fragment adherent method)

1. Take about 10 cm of fresh umbilical cord and place it in the culture dish under sterile condition; Soak and wash

three times with PBS containing penicillin-streptomycin to remove the blood; Use tweezers to scrape the residual blood from the umbilical vein along the outer surface of the umbilical cord to both sides; When the tissue

becomes clean and translucent, cut the umbilical cord tissue into 2 cm segments.

2. Longitudinally dissect each segment of the umbilical cord with ophthalmic scissors; Remove the blood

vessels (1 umbilical vein and 2 umbilical arteries); Cut the tissue into segments of about 2 mm3 or peel off the

gel-like tissue (Wharton's jelly) and cut into minced pieces before placing it into a centrifuge tube.

3. Umbilical cord tissue: Evenly seed the tissue segments in the culture dish (maintaining a distance of 0.5 cm

between the segments); Gently add one drop of MSC medium to each tissue segment; Place the culture dish in

the incubator at 37℃ and 5% CO2 for culture; Gently supplement the medium along the side wall of the culture

dish after 2 hours.

Wharton's jelly: Add MSC medium into the centrifuge tube and resuspend the tissue fragments in the complete

medium; Seed the tissue fragments in the culture dish and gently shake the culture dish to evenly distribute the

tissue fragments; Place the culture dish in the incubator at 37℃ with 5% CO2 for culture.

4. Monitor the cell growth condition in 1-3 days; Change the medium for the first time after 7 days, and then

change the medium once every 3-4 days; When the cells confluence reaches 80%-90% (about 12-15 days),

digest and passage with trypsin; Transfer the tissue to a new culture dish and repeat the culturing protocol as

mentioned above.

< Isolation and culture of human umbilical MSCs >

< Isolation and culture of bone marrow MSCs >

www.sinobiological.com 3

umbilical arteries

umbilical cord blood umbilical vein

Wharton’ Jelly

Umbilical cord

structure

Cultivation of umbilical

cord tissue

Cultivation of

Wharton's jelly

Expansion of MSCs

MSC cell Recovery

1. Take out the cryopreserved MSC cells from liquid nitrogen; Quickly place the cryovial into a 37℃ and monitor

closely; When the cells are mostly thawed, immediately remove the cryovial from the water bath.

2. Use a pipette to gently aspirate all the contents from the cryovial into a sterile 15 mL centrifuge tube.

3. Slowly add the MSC culture medium preheated to 37℃ to the centrifuge tube. Avoid adding the medium all at

once to prevent a reduction in cell viability due to osmotic pressure turbulence; Gently Pipette twice to resuspend the cells.

4. Centrifuge at 100-200 g for 5 minutes, then aspirate and discard the supernatant; Add MSC culture medium

to resuspend the cells, adjust the cell density, and seed them in a culture flask at 5,000-6,000 cells/cm2; Place

the flask in an incubator at 37℃ with 5% CO2 for culture.

5. Change the culture medium every 2-3 days; When the cells confluence reaches 80%-90%, passage the cell

accordingly.

MSC cell subculture

1. Aspirate and discard the culture medium in the culture flask; Wash the cells by DPBS (without calcium and

magnesium ions); Aspirate and discard the DPBS, and add an appropriate amount of trypsin preheated at 37℃

(e.g., about 3 mL for a T25 culture flask); Incubate at 37°C until the cells are completely dissociated (about 3-5

minutes).

2. Add the culture medium and gently resuspend the cells; Collect the mixture into a centrifuge tube.

3. Centrifuge at 100-200 g for 5 minutes; Aspirate and discard the supernatant, and resuspend the precipitate

with the culture medium; Adjust the cell density and seed the cells in the culture flask at 5,000-6,000 cells/cm2;

Change the culture medium every 2-3 days.

When MSC grows slowly, it is recommended to add some cytokines to promote growth.

For example: 20 ng/mL EGF (cat#: 10605-HNAE/50482-MNCH), 20 ng/mL FGF2 (cat#: 10014-HNAE/50037-M07E),

10 ng/mL PDGF-BB (cat#: 10572-HNAE), and10 ng/mL TGF-β (cat#: 10804-HNAC).

MSC cell cryopreservation

1. Prepare the cell cryopreservation solution: For 1 mL of the cryopreservation solution, use 0.9 mL of MSC medium

plus 0.1 mL of dimethyl sulfoxide (DMSO) (Final concentration 10%).

2. Resuspend the harvested cell pellet (recommended cell cryopreservation density at 2×106 cells/mL) using 1

mL of the cell cryopreservation solution; Gently Pipette to ensure even distribution of the cells, and transfer them

to a cryotube pre-cooled at 4℃.

3. Place the cell cryotube into a cryobox containing isopropanol; Place the cryobox in a -20°C freezer for 2 hours

and in a -80°C freezer overnight; Finally, transfer the cryotube to a liquid nitrogen container for long-term

storage.

北京义翘神州科技股份有限公司 www.sinobiological.com 4

1. Prepare the cell culture dishes coated with 0.1% gelatin. (Add an appropriate amount of gelatin coating solution to the culture dish to cover the bottom of the well plate. Incubate for 30 minutes, then aspirate and discard

the coating solution.) Prepare the complete medium for inducing osteogenic differentiation (α-MEM, 10% FBS,1%

P/S, 0.05 mM Ascorbic acid, 10 mM β-Glycerophosphate sodium, 100 nM Dexamethasone).

2. Obtain the MSCs with cell confluence reaching about 85%, and digest with trypsin. After the cells are dissociated, add complete medium to stop the digestion, and centrifuge at 300 g for 5 minutes.

3. Adjust the cell density to 5×103 cells/cm2 and seed in the treated 6-well plate. Add 2 mL of MSC complete

medium to each well. When the cell confluence reaches 60%, replace the growth medium with the complete

medium for inducing osteogenic differentiation and initiate the osteogenic differentiation. Meanwhile, culture

undifferentiated MSC in the standard complete medium (α-MEM, 10% FBS) as a control.

4.Culture the cells in an incubator at 37℃ and 5% CO2 for 2-4 weeks. Change the osteogenic medium twice a

week.

5. Alkaline Phosphatase (ALP) Staining: On day 7 since osteogenic induction, apply the alkaline phosphatase kit

to evaluate the ALP activity of the cells.

6. Alizarin red staining: On day 21 since osteogenic induction, aspirate and discard the complete medium for

osteogenic differentiation in the 6-well plate. Wash with PBS 1-2 times. Add 4% paraformaldehyde solution

(enough to cover the cell surface) and fix the cells for 30 minutes. Aspirate and discard the solution. Wash with

PBS for 1-2 times. Add an appropriate amount of alizarin red working solution (enough to cover the cells) and

stain at room temperature for 5-10 minutes. Aspirate and discard the staining solution. Wash with PBS 1-2 times.

Observe the osteogenic staining effect under the microscope.

Adipogenic Differentiation of MSCs

1. Prepare cell culture dishes coated with 0.1% gelatin. (Add an appropriate amount of gelatin coating solution

to the culture dish to cover the bottom of the well plate. Incubate for 30 minutes, aspirate and discard the coating solution.) Prepare the induction adipogenic differentiation medium A (High-glucose DMEM, 10% FBS, 1% P/S,

1 µM Dexamethasone, 0.5 mM IBMX, 100 µM Indomethacin, and 10 µg/mL Insulin) and induction adipogenic

differentiation medium B (High-glucose DMEM, 10% FBS, 1% P/S, 10 µg/mL Insulin).

2. Obtain the MSCs with cell confluence reaching about 85%, and digest with trypsin. After the cells are dissociated, add complete medium to stop the digestion, and centrifuge at 300 g for 5 minutes.

Osteogenic Differentiation of MSCs

Reference for alizarin red staining

100μm 100μm 100μm

Bone Marrow MSCs

DOI : 10.3389/fbioe.2023.1152207

Adipose MSCs

DOI : 10.3389/fbioe.2023.1152207

Umbilical Cord MSCs

DOI: 10.1111/jdi.14077

Multilineage Differentiation of MSCs

www.sinobiological.com 5

3. Adjust the cell density to 2-3×104 cells/cm2 and seed in the treated 6-well plate. Add 2 mL of MSC complete

medium to each well. When the cell confluence reaches 90-100% (the cell oversaturation situation is more beneficial to adipogenic differentiation), replace the growth medium with induction adipogenic differentiation

medium A and initiate the adipogenic differentiation. After 2 days of induction, aspirate and discard the

medium, treat the cells with induction adipogenic differentiation medium B for 1 day and then change the culture

medium to medium A for further induction. Repeat this alternating cycle of culture until day 14. Perform cell morphological observation. After sufficient and appropriately sized lipid droplets appear, conduct Oil Red O staining.

4.Oil Red O staining: Take 0.5 g of Oil Red O dry powder and add to 100 mL of isopropanol. Fully dissolve, keep it

sealed and protected from light at 4°C for storage as the stock solution. Take the stock solution and mix with

distilled water at a ratio of 3:2. Filter it with qualitative filter paper to avoid precipitation. Use the filtrate as the

working solution.

Aspirate and discard the medium. Wash with PBS for 1-2 times. Add 4% paraformaldehyde solution (enough to

cover the cells), and fix the cells for 30 minutes. Aspirate and discard the solution. Wash with PBS for 1-2 times.

Add an appropriate amount of the working solution of Oil Red O (e.g., 1 mL per well for a six-well plate), and stain

for 15-30 minutes. Aspirate and discard the staining solution. Wash with PBS/distilled water 3 times. Observe the

adipogenic staining under a microscope.

1. Prepare the induction chondrogenic differentiation medium (High-glucose DMEM, 20 ng/ml TGF-β3 (cat#:

10434-H01H), 100 nM Dexamethasone, 1% ITS, 50 µg/ml Ascorbic Acid, 1 mM Sodium Pyruvate, 40 µg/ml Proline).

2. Obtain the MSC with cell confluence reaching 80%-90%, digest with trypsin. After the cells are dissociated, add

complete medium to stop the digestion. Acquire 3-4×105 MSC cells and transfer them to a 15 mL centrifuge tube,

and centrifuge at 200 g for 5 minutes.

3. Aspirate and discard the supernatant. Gently add 2 mL of the chondrogenic differentiation induction medium

along the wall of the centrifuge tube (Ensure that the cells are not disrupted. If the cells are disrupted, centrifuge

again). Carefully loosen the cap of the centrifuge tube half-twist to facilitate gas exchange and place it in a 37°C,

5% CO2 incubator for culture.

4. After static culture for 24-48 hours, gently tap the bottom of the centrifuge tube to make the spherical cell

aggregates detach from the wall and suspend in the medium. Gently replace the differentiation medium and

place in a 37°C, 5% CO2 incubator for culture. From then on, replace the fresh differentiation medium every 2-3

days. Gently tap the bottom of the centrifuge tube in the morning and evening every day to encourage the cartilage spheres to become more spherical.

5. The diameter of the cartilage spheres gradually increases during the induction process, and the surface

becomes smooth and gelatinous. After continuous induction for 14-24 days, when the diameter of the cartilage

spheres reaches 1.5-2 mm, they can be fixed, sectioned, and stained with Safranin O, Alcian Blue, or antibodies to

identify chondrocytes.

Chondrogenic Differentiation of MSCs

Reference for oil red O staining

Bone Marrow MSCs

DOI : 10.3389/fbioe.2023.1152207

Adipose MSCs

DOI : 10.3389/fbioe.2023.1152207

Umbilical Cord MSCs

DOI: 10.1111/jdi.14077

100μm 50μm 100μm

www.sinobiological.com 6

https://cdn1.sinobiological.com/web2024/msc18.png

https://cdn1.sinobiological.com/web2024/msc32.jpg

https://cdn1.sinobiological.com/web2024/msc33.jpg

https://cdn1.sinobiological.com/web2024/msc4.png

Reference for alcian blue staining

Table 1. Surface markers of MSCs

Table 1. Surface markers of MSCs for additional test in clinical

Bone Marrow MSCs

DOI : 10.3389/fbioe.2023.1152207

Adipose MSCs

DOI : 10.3389/fbioe.2023.1152207

Umbilical Cord MSCs

DOI: 10.1155/2022/9124277

100μm 100μm 100μm

www.sinobiological.com

Identification Markers of MSCs

Surface markers of MSCs

The International Society for Cell Therapy (ISCT) has proposed a set of criteria for analyzing MSCs based on the

characteristics of cultured cells in vitro: (1) Adherence during cultivation; (2) Specific identification markers of

MSCs (Table 1); (3) The ability of in vitro trilineage differentiation.

According to the suggestions in the \"Quality Inspection Specifications for Mesenchymal Stem Cells for Clinical Research (Draft for

Comment)\", the clinical application of MSCs requires additional testing for CD44 and CD166 (Table 2).

Target CAT# HMSC03 HMSC07

CD105 10149-MM13-A √ √

CD73 10904-MM07-P √ √

CD90 16897-MM10-C √ √

CD45 10086-MM05-F √ √

CD34 68035-XM01-F √ √

CD14 or CD11b 10073-MM06-F √ √

CD79α or CD19 11880-MM17-F √ √

HLA-DR 68038-XM01-F √ √

Positive markers

of MSCs (≥95%)

Negative markers

of MSCs (≤2%)

Target CAT HMSC03 HMSC07

CD44 12211-MM02-F √ √

CD166 10045-MM03-A √

Positive markers

of MSCs (≥95%)

7

6. Alcian Blue staining: Gently wash the cartilage spheres with PBS for 1-2 times. After fixing with neutral formaldehyde or formalin, embed in paraffin and section. Dewax the paraffin sections, stain with Alcian Blue staining solution for 30 minutes, rinse with running tap water for 2 minutes, and rinse with distilled water for 30 seconds to stop

the staining, check the staining under the microscope.

北京义翘神州科技股份有限公司

Identification by flow cytometry

The Human MSC Analysis Kit, catalog numbers HMSC03 and HMSC07, are specialized kits for identifying human

mesenchymal stem cells. They offer a comprehensive range of test items, are simple and easy to use, and

provide accurate and reliable results.

1. Collect MSCs when cell confluence reaches approximately 90%. Discard the culture medium, wash the cells

three times with PBS, and then digest with trypsin. Once the cells are dissociated, add complete culture medium

to stop the digestion, and centrifuge at 300 g for 5 minutes.

2. Resuspend the cells at a concentration of 1×107

-2×107

cells/mL. Add 50 µL of the cell suspension to each test

tube.

3. Using the HMSC07 kit as an example, prepare 8 test tubes. Add 20 µL of each antibody to the respective test

tube, gently mix, and incubate in the dark at 4℃ for 20 minutes.

(1) PE mouse anti-human CD73 (20 µL)：Flow cytometry compensation.

(2) PerCP mouse anti-human CD90 (20 µL)：Flow cytometry compensation.

(3) APC mouse anti-human CD105 (20 µL)：Flow cytometry compensation.

(4) FITC mouse anti-human CD44 (20 µL)：Flow cytometry compensation and detection of the expression of

CD44.

(5) Positive cocktail-1 (20 µL) + FITC negative cocktail (20 µL)：Detection of the expressions of positive markers

CD105, CD73, CD90 and negative markers CD45, CD34, CD14, CD19, HLA-DR.

(6) Positive isotype control cocktail (20 µL) + FITC Mouse IgG1 isotype control (20 µL)：The isotype control

antibodies of positive markers CD105, CD73, CD90,CD44 and negative markers.

(7) APC mouse anti-human CD166 (20 µL)：Detection of the expression of CD166.

(8) APC Mouse IgG1 isotype control (20 µL)：The isotype control antibody of the positive marker CD166.

4. Wash with PBS three times, resuspend the cells with 300-500 µL PBS, and analyze the cells with a flow cytometer.

Cultured hMSCs from umbilical cord are analyzed by MSC07. Cells demonstrate negative expression of CD45, CD34, CD14,

CD19, HLA-DR (figure A) as well as positive expression of CD73 (Figure B), CD90 (Figure C), CD105 (Figure D), CD44 (Figure E),

CD166 (Figure F). The blue lines are cells with isotype antibodies (tube 6). The red lines are cells with antibodies targeting to

marker (tube 5).

Flow cytometry of hMSCs by Human MSC Analysis Kit (Cat#: HMSC07)

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Disclaimer: This experimental operation process is only for sharing experience and providing a reference for

scientific research work. We strive to ensure the accuracy and reliability of the content. However, there are uncertainties and complexities in scientific research experiments. If the experiment based on this process fails to

achieve the expected results or fails, Sino Biological Inc. assumes no responsibility. When using it, please combine

the actual situation, make professional judgments and operate with caution. Moreover, this process is only for

scientific research and cannot be used for commercial or illegal purposes. The final right of interpretation belongs

to Sino Biological Inc. Hereby declared.

Featured Cytokines for MSC Culture

Cytokines CAT# Species Expression Host Purity Endotoxin

10014-HNAE Human E. coli

50482-MNCH Mouse HEK293 Cells

TGF beta 1 10804-HNAC Human, Rhesus,

Cynomolgus, Canine

CHO Stable

Cells

50437-MNAY Mouse Yeast

50159-MNAB Mouse

BaculovirusInsect Cells

IGF1

10598-HNAE Human E. coli

PDGF-BB 10572-HNAE Human E. coli

VEGF-A

10008-HNAH Human HEK293 Cells

basic FGF/FGF2

50037-M07E Mouse E. coli

EGF

10605-HNAE Human E. coli

≥ 95% SDS-PAGE < 10 EU/mg

≥ 95% SDS-PAGE

≥ 95% SEC-HPLC

≥ 95% SDS-PAGE

≥ 95% SEC-HPLC

> 95% SDS-PAGE < 1 EU/µg

≥ 95% SDS-PAGE

≥ 95% SEC-HPLC

> 95% SDS-PAGE < 1 EU/µg

> 95% SDS-PAGE

> 95% SEC-HPLC

≥ 90% SDS-PAGE

≥ 90% SEC-HPLC

≥ 95% SDS-PAGE

≥ 90% SEC-HPLC

> 95% SDS-PAGE

< 1 EU/µg

<10 EU/mg

< 50 EU/mg

< 1 EU/µg

< 10 EU/mg

< 5 EU/mg

9

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