HiFi™ Wharton’s Jelly Mesenchymal Stem Cells (HWJ-MSC) PRODUCT CODE: CL001

Product WarrantyHiFi™ cells are performance tested using HiMedia media and

reagents. Users must ensure proper handling, storage and use of

the product. We DO NOT GUARANTEE the performance of the cells if protocols and products other than those mentioned in this sheet are used. We are not liable for any damage to the culture arising from improper storage or mishandling.

Initial Handling upon ReceiptFor any damage to the product upon receipt, report it

immediately to the HiMedia marketing representative or technical

team.

For any difficulty while handling or processing the cells please

contact HiMedia technical team immediately for flask and within

one month for cryovial after delivery:

022-61169797 / 8767552556 or atc@himedialabs.com

Check List• Proliferating Cells (Refer Table 1): Cells are supplied in flasks

with closed (non-vented) caps. Loosen one thread of the cap

only after medium change. Use sterile disposable tips and

serological pipettes. Avoid using autoclaved tips and serological

pipettes.• Cryopreserved Cells (Refer Table 2): Immediately transfer the

vial to a liquid nitrogen tank upon receipt using forceps. DO NOT

hold the vial in hand. DO NOT store the cells at -80⁰C.

Common MistakesStem cells and primary cells are more sensitive and delicate as compared to the robust continuous cell lines hence, require extreme precautions while handling, subculturing and thawing. Following handling methods can lead to loss of cell viability, alteration in morphology and loss of attachment of cells.

1. Exposing the cryopreserved cells during thawing to warm

temperatures (37oC) for more than 90 - 120 secs2. Not changing medium within 2-3 hours after thawing

3. Sub-culturing after the cells reach 100% confluence

4. Exposing the cells to trypsin for more than the recommended

time during sub-culturing

Product Description and ContentsHiFi™ Wharton’s Jelly Mesenchymal Stem Cells (HWJ-MSC) are

isolated from human umbilical cords collected post partum.

Wharton’s jelly is the gelatinous connective tissue from umbilical

cord and is a rich source of multipotent stem cells.

HWJ-MSC are Passage 2 cells supplied frozen with density of not

less than 0.5 x 106 cells per vial or as proliferating cells in a T-25

culture flask. Each lot of cells is from a single donor and undergoes

growth kinetic studies, stem cell marker analysis and functional

analysis of multilineage differentiation. Cells are maintained in

antibiotic-free conditions prior to supply.

Source : Human

Tissue : Umbilical Cord (Wharton's Jelly)

Type of Cells : Mesenchymal

Growth Characteristics : Adherent

Products Required But Not Supplied1. Growth medium Code

HiMesoXL™ Mesenchymal Stem Cell Expansion Medium [or]HiMesoXL™ Mesenchymal Stem Cell Expansion Medium

AL512

AL519 (Reduced Serum)

2. Media Supplements Code

Mesenchymal Stem Cell Tested Fetal Bovine Serum (FBS)

RM10832, RM10845 RM10846, RM10938

Antibiotic-Antimycotic Solution 100X [or] Gentamicin- Amphotericin B solution 1000X

A002

A031

3. Reagents for Sub-culture Code

Dulbecco’s Phosphate Buffered Saline (DPBS)

TL1006

Trypsin/EDTA Solution 1X TCL007Trypan Blue 0.5% solution TCL005

4. Stem Cell Freezing Medium CodeCryoXL™ Stem Cell Freezing Medium

TCL107

PRODUCT INFORMATION

Trypsin inhibitor from soyabean TCL068

Table 1 : Storage and Handling of Proliferating Cells• Proliferating cells are supplied in a T-25 closed (non-vented cap) flask at room temperature.• The flasks are filled to capacity with transport medium and the neck is sealed with parafilm.• Each flask is individually packed in a plastic bag to contain leakage, if any.

Key Points to Remember

Time

Required

(approx.)

Initial Handling upon Receipt

• Work in areas designated foruse Human origin materialBiosafety Level II (BSL II)

• Wear protective clothing andgloves

→ CHECK• Leakage during transport

Flask leaking or broken

Discard in bio-hazardous waste, if required

Intact flask Remove the flask from plastic bag

→ CHECK• Cell morphology and

confluence• Microscopic visibility of the

cells is hampered due to flaskfilled to capacity

DO NOT discard the medium!120 secs

→ CHECK• Incubate the cells for 3-4 hrs to

allow floating cells to reattachto the surface

DO NOT loosen one thread of the cap! 3 hrs

• After incubation, remove the medium and discard

• Add 5-7 ml fresh medium inthe flask

• At this stage cells will be clearlyvisible under the microscope

180 secs

• Incubate at 37oC and 5% CO2 Loosen one thread of the cap!1-3 days

YOUR CELLS ARE READY TO SUB-CULTURE

Table 2 : Storage and Handling of Cryopreserved Cells• Cryopreserved cells are supplied in liquid nitrogen dry vapour shipper (-150oC to -130oC).• Upon receipt, immediately transfer the vial to the vapor phase of liquid nitrogen tank.• Store it in the tank until further use. Cells must be processed at least in a BSL II hood.

Key Points to Remember

Time

Required

(approx.)

1. Preparation of Culture Vessel

a. Add 5ml of complete mediumto a T-25 flask

Preparation of complete medium AL512 (500 ml) + FBS (55ml) + A002 (5.5 ml) [or] AL519 (Part A 500 ml) + (Part B 11.3 ml) + A002 (5 ml)

For details, refer Technical Data Sheet of AL512 or AL519

60 secs

b. Place the flask at 37°C toequilibrate the medium

2. Thawing Procedure

a. Remove cryovial from theliquid nitrogen tank/ shipperwearing appropriateprotective gear

Thawing should be AS FAST AS POSSIBLE to minimize cell damage

b.DO NOT hold the vial in water bath for more than 90-120 secs

AVOID getting water upto the cap of the vial

90-120secs

c. Disinfect the vial by swabbingthoroughly with 70% isopropylalcohol

10 secs

d. Add the cell suspension dropby drop to the T-25 flaskcontaining the pre-warmedcomplete medium. Keepswirling the flask while addingthe cell suspension

Dropwise addition is required to prevent the cells from stress induced by exothermic reaction

30-60 secs

30 mins

Make sure water bath is set at 37⁰C before starting the thawing procedure

Immediately thaw the vial partially by holding in a water bath at 37°C

Table 2 : Storage and Handling of Cryopreserved Cells• Cryopreserved cells are supplied in liquid nitrogen dry vapour shipper (-150oC to -130oC).• Upon receipt, immediately transfer the vial to the vapor phase of liquid nitrogen tank.• Store it in the tank until further use. Cells must be processed at least in a BSL II hood.

Key Points to Remember

Time

Required

(approx.)

e. Cap the flask and shake gentlyto ensure proper mixing anduniform distribution of cells inthe medium

10 secs

3. Incubation

a. Incubate the cells at 37°C and5% CO2

Check for cell attachment in 2-3 hrs 2-3 hrs

b. If more than 70-80% cells areattached, replace the mediumwith fresh medium

Medium change after 2-3 hours is mandatory to remove traces of DMSO

If cells have not attached, centrifuge the cell suspension at 1000 rpm for 7-8 mins and resuspend in fresh medium

60-120secs

c. Incubate the cells at 37°C and5% CO2

3-5 days

YOUR CELLS ARE READY TO SUB-CULTURE

4. Maintenancea. Monitor the cells every day

b. Change the medium everyalternate day

c. Sub-culture, once cells reach70 - 80% confluence

7-8 min

Use the recommended freezing medium for cryopreservation of cellsDO NOT allow cells to reach 100% confluency before sub culture or cryopreservationIn case of reduced serum or serum free media , use trypsin inhibitor solution (TCL068) for neutralisation of Trypsin during subcultureUsage of just medium for neutralisation will result in inefficient neutralisation and will stress the cells resulting in reduced viability and cell death

Table 3 : Sub-culture• HWJ-MSC can be sub-cultured at a seeding density of 5000-10,000 cells/cm2.• Sub-culturing ratios can vary from 1:2 - 1:5• A confluent T-25 flask of HWJ-MSC yields 1.0 x 106 cells

Key Points to Remember

Time

Required

(approx.)

a. Aspirate entire medium anddiscard

DO NOT disturb the monolayer60 secs

b. Wash the cells with 2-3 mlDPBS to remove residualmedium

c. Aspirate off the DPBS anddiscard

Prior to use, make sure that Trypsin- EDTA solution is equilibrated to room temperature

60 secs

d. Add 0.5 ml pre-warmedTrypsin-EDTA solution

Gently rock the flask to ensure complete coverage of the Trypsin-EDTA solution over the cells

e. Incubate the flask at 37°C Exposing the cells to Trypsin-EDTA for longer time leads to loss of cell viability

30-60 secs

f. Microscopically monitor theflask

g. When the cells start roundingup, gently tap the flask toensure complete detachmentof cells

15 secs

h. To neutralize action oftrypsin add 3 ml of completemedium, if AL512 is used

i. Pipette gently to get ahomogenous mixture of cells

Vigorous pipetting will stress the cells 60 secs

j. If reduced serum medium AL519 is used, add 0.5 ml Soyabean Trypsin Inhibitor Solution (TCL068). Centrifuge the cell suspension at 1000 rpm for 10 mins. Discard supernatent and resuspend pellet in fresh 3 ml of complete medium by pipetting.

Table 4 : Seeding Density

Flask Recommended Seeding Densitiy No. of Cells Per Flask Volume of Medium (ml)

T-255000 cells/cm2 0.125 x 106 5 - 7

10,000 cells/cm2 0.25 x 106 5 - 7

These are recommended seeding densities from literature and our studies. Higher seeding densities do not cause any harm to the cells and reduce the

required population doublings per passage. Lower seeding densities may cause cells to lose viability, detach during culture and in general take more

population doublings to reach confluence.

DO NOT refrigerate cells after splitting

Seed immediately

10-15

minsk.

Count cells using hemocytometer

l. Seed at recommended seeding density in a new flask containing fresh complete medium

Refer to Table 4

Table 3 : Sub-culture• HWJ-MSC can be sub-cultured at a seeding density of 5000-10,000 cells/cm2.• Sub-culturing ratios can vary from 1:2 - 1:5• A confluent T-25 flask of HWJ-MSC yields 1.0 x 106 cells

Key Points to Remember

Time

Required

(approx.)

Count cells using hemocytometer

m. Incubate in a humidifiedincubator at 37ºC and 5% CO2

48 hrs

Maintenance

a. Monitor the cells every day

b. Change the medium everyalternate day

c. Sub-culture once cells reach70 - 80% confluence

10-15

Growth Performance Assay No. of viable cells/vial : NLT 500,000 cells/vial

Percentage viability : NLT 80%

Total no. of population doublings : NLT 15

Marker Analysis Assay By Flow Cytometry

This data has been generated on 8 parameter 3 laser

Partec CyFlow® Cube 8 Flow Cytometer and is specific for

representative batch of HWJ-MSC.

Positive Markers (>95% events) CD90, CD105

Negative Markers (<5% events) CD34, CD45

Di�fferential Potential Adipogenic : Positive

Osteogenic : Positive

Chondrogenic : Positive

Virus TestingHIV (PCR): Not detected

Hepatitis B Virus (PCR): Not detected

Hepatitis C Virus (PCR): Not detected

WarningThis product is intended for research use only. Not for

animal, human therapeutic or diagnostic use.

Product contains human origin material and should be treated as

potentially infectious. Even if the cells provided have been screened

for viral and bacterial pathogens, human cells may harbor other

known or unknown agents which might pose a health hazard.

Universal handling precautions applicable to biological samples

must be applied as recommended in the CDC-NIH manual.

Quality Control

Sterility TestingMycoplasma (PCR): Not detected

Bacteria, Fungi and Anaerobes as per USP: Not detected

Table 5 : Related Products

Product Name Code Packing

Fetal Bovine Serum, MSC Tested EU Approved, Sterile Filtered

RM10832-100ML RM10832-500ML

100 ml500 ml

Fetal Bovine Serum, MSC Tested USDA Approved, Sterile Filtered

RM10845-100ML RM10845-500ML

100 ml500 ml

Fetal Bovine Serum, MSC Tested Australia Origin, Sterile Filtered

RM10846-500ML100 ml500 ml

HiAdipoXL™ Adipocyte Differentiation Medium AL521-100ML 1x100 ml

HiOsteoXL™ Osteocyte Differentiation Medium AL522-100ML 1x100 ml

HiChondroXL™ Chondrocyte Differentiation Medium AL523-100ML 1x100 ml

EZstain™ Adipocyte Staining Kit CCK013-1KT 1 Kit

EZstain™ Osteocyte Staining Kit CCK030-1KT 1 Kit

EZstain™ Chondrocyte Staining Kit CCK029-1KT 1 Kit

Disclaimer :

User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and other related

HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the best of our knowledge true and

accurate. HiMedia™ Laboratories Pvt. Ltd. reserves the right to make changes to specifications and information related to the products at any time. Products are not

intended for human or animal diagnostic or therapeutic use but for laboratory, research or further manufacturing use only, unless otherwise specified. Statements

contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd.

A-516, Swastik Disha Business Park, Via Vadhani Indl. Est. LBS Marg, Mumbai - 400 086, India. Customer Care No. : 022-61471919 | Email : atc@himedialabs.com

Revision: 2/ 2018