USER GUIDE

Activated Myofibroblastic Hepatic Stellate Cells
Catalog Numbers HMFHSC
Pub. No. MAN0018349 Rev. 2.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
IMPORTANT! Cryopreserved stellates should be kept at all times in the vapor phase of liquid nitrogen. The product shall only be removed
when it is needed and thawed immediately after removal from the liquid nitrogen container. Cryopreserved hepatic stellates must be
stored at all times in the vapor phase of liquid nitrogen, not submerged. The product should only be removed when it is to be thawed andcultured, which should occur immediately after removal from liquid nitrogen storage.

Product description
Activated Myofibroblastic Hepatic Stellate Cells (MF-HSCs) are produced from isolated pure quiescent human stellate cells (q-HSCs). The
q-HSCs are activated to become MF-HSCs through a process of passaging and maintence in cell culture media. These activated MF-HSCs
are ideal for building hepatic co-culture with primary hepatocytes to study in vitro disease models such as hepatic fibrosis. The cells are
obtained from a single donor and provided in a cryopreserved format identical to single donor plateable hepatocytes. Additionally, every
lot of cells is tested for cell viability, plating efficiency, morphology, purity based on α-smooth muscle actin (α-SMA) expression, andactivation in response to Transforming Growth Factor-β1 (TGF-β1).

Contents and storage
Contents
Activated Myofibroblastic Hepatic
Stellate Cells

AmountShipping

Storage

1 mL (>1.0 × 106 cells)

Liquid nitrogen (vapor phase)

Liquid nitrogen (vapor phase).

Required materials not supplied

Product specifications

Unless otherwise indicated, all materials are available throughthermofisher.com.
Item
Nunc™ 15 mL & 50 mL Conical Sterile
Polypropylene Centrifuge Tubes
RNase-Free Microfuge Tubes
Collagen I, Coated Plate, 6- or 24‑well

Description

Details

Form

CryopreservedSpecies

HumanQuantity

1 CryoVial

AM12400

Donor source

1 donorA1142801, A1142802

Product size

1 mL

Source339651, 339653

DPBS, no calcium, no magnesium14190144

StemPro™ Accutase™ Cell Dissociation
Reagent

A1110501

Trypan Blue Solution, 0.4%15250061

Advanced DMEM

12491015

GlutaMAX™ Supplement

35050061

Fetal Bovine Serum, certified, US origin

16000044

Penicillin-Streptomycin (10,000 U/mL)

15140122

For Research Use Only. Not for use in diagnostic procedures.

Number of cells

1 × 106 cells

Procedural guidelines• Cryopreserved cells should be thawed quickly in a 37°C
water bath.
• Once thawed, cryopreserved MF-HSC must be used
immediately and cannot be refrozen.

Prepare activated/myofibroblastic stellate cell
plating media (MF-Plating Media)
Aseptically mix the following media and reagents:
Reagent

Amount

11. Place the culture plate in a 5% CO2 incubator maintained at37°C. In a back-and-forth and side-to-side motion, shake theplate to evenly and gently distribute the cells in the culturewell.
12. Culture the cells undisturbed for at least 12–16 hours before
using them for experimentation.

Advanced DMEM

500 mL

GlutaMAX™ Supplement

5 mL

Change media

Fetal Bovine Serum, certified, US origin (finalconcentration ~10%)

50 mL

When culturing MF-HSCs longer than three days, the media
should be changed once every three days.

Penicillin-Streptomycin (1X finalconcentration)

5 mL

1. Warm the plating media and DPBS in a 37°C water bath.

MF-Plating Media can be saved in 4°C, protected from light, up toone month.

Thaw and plate cells
1. Warm up the plating media prepared above in a 37°C water
bath for at least 30 minutes before thawing cryopreservedcells.
2. Decontaminate the external surfaces of the stellate plating
media bottle with 70% ethanol and transfer it to a biosafetycabinet.
3. In a 15-mL centrifuge tube, add 9 mL of activatedmyofibroblastic stellate cell plating media from Step 1.
4. Remove the vial of cells to be thawed from liquid nitrogenand rapidly thaw by placing in a 37°C water bath with gentle
agitation for 1–2 minutes (or once a sliver of ice is left in thetube).
Complete thawing can be detrimental to the cell viability.
5. Decontaminate the external surface of the cryovial with
70% ethanol. Transfer the vial into the biosafety cabinet andpromptly transfer the contents of the vial to a15-mL centrifuge tube containing 9 mL of plating media.
6. Centrifuge the 15-mL centrifuge tube containing the stellate
cells at 500 × g for 5 minutes at room temperature.
7. Carefully aspirate the supernatant.
Make sure not to disturb the pelleted stellate cells.

2. In the biosafety cabinet, aspirate the media from the culture
plate. Rinse the stellate cells with pre-warmed DPBS with
1 mL per well for the 24-well plate or 4 mL per well for the6-well plate.
3. Aspirate DPBS away from the plated cells.
Make sure not to disturb the plated cells.
4. Add 0.5 mL of plating media per well for the 24-well plate or
2 mL of plating media per well for the 6-well.
5. Return the culture plate to the 5% CO2 incubator maintained
at 37°C.

IMPORTANT! We do not recommend further passaging of

hepatic stellate cells, because further passaging will reduce
the level of activation of the HSCs.

Related products
Unless otherwise indicated, all materials are available throughthermofisher.com. MLS: Fisher Scientific (fisherscientific.com) orother major laboratory supplier.
ItemSource

Human Plateable Hepatocytes, Induction
QualifiedHMCPIS

Human Plateable Hepatocytes, TransportQualifiedHMCPTS

Spheroid Qualified Human Hepatocytes

HMCPSQ

8. Resuspend the cells in 1 mL of pre-warmed stellate cell
plating media by gently pipetting up and down.
9. Take 10–20 μL of the resuspended cells and count them using
hemocytometer or any other counting device.
10. Plate the cells in collagen I coated plates by diluting toapproximately 10,000 cells/well for a 24-well plate or
40,000 cells/well for a 6-well plate to achieve a 30–
40% confluency after 24-hours.
We recommend that you seed the cells in 500 μL plating
media per well for 24-well plates and 2-mL plating media perwell for 6-well plates.

2

Activated Myofibroblastic Hepatic Stellate Cells User Guide

Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms andConditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact
Life Technologies at www.thermofisher.com/support.

Life Technologies Corporation | 3175 Staley Road | Grand Island, NY 14072
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
1 November 2019