Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Summary

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Abstract

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.

hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents.

We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

Introduction

Cells derived from perinatal sources, such as the placenta, placental membranes, umbilical cord and amniotic fluid have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine^1,2. The reason for this interest is that these cell types all possess some degree of plasticity and immunomodulatory capability³, properties that are fundamental to their potential therapeutic applications.

hAECs are a heterogeneous epithelial population that can be derived from term or pre-term amnion membrane⁴, providing an abundant potential source of regenerative cellular materia....

Protocol

NOTE: Placentae should be collected from singleton healthy pregnancies, with a preference for term elective caesarean sections. Written, informed consent should be given for the collection of their placenta. Your relevant human research ethics committee should approval all collection and use of human tissues.

1. Isolation of Amnion Epithelial Cells

1. Place the placenta onto a sterile surface within a class II biological safety cabinet.
2. Using sterile hanks balanced salt solution (HBSS), wash as much blood as possible from the placenta surface and membranes.
3. With the placenta placed wit....

Results

When this procedure is followed correctly, an average yield of 120 million hAECs should be expected, with a typical range of 80-160 million cells. From these yields, an average viability of 83 ± 4% can be expected. The increased average yield and slightly lower viability in the clinical method may be due to higher trypsin activity than the animal-derived product, and perhaps also due to the lack of serum proteins. Isolated hAECs have an average cell surface profile of 92% EpCAM positive cells with <1% CD90, CD105 mese.......

Discussion

There are several critical parameters that can have a significant impact in the success of this methodology. Storage of the placenta or amnion for up to 3 hr before isolation of hAECs may be desirable for logistic or scheduling purposes, however it is recommended that the tissue is processed as soon as possible. If tissue is to be stored, it is recommended that storage be performed following dissection and washing of the amnion membrane. Amnion can be stored in sterile HBSS containing antibiotics at 4 °C, however ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collection Kit
Stripping tray	Fisher Scientific	13-361B
Liberty Dressing Forcep, pointed 13 cm	Fisher Scientific	S17329
Scissors - Sharp/Blunt Straight	Fisher Scientific	NC0562592
Sterile latex gloves	Fisher Scientific	19-014-643
Protective Apparel
Protective Apparel (Gown)	U-line	S-15374-M
Isolation gowns	U-line	S-15374-M
Sterile latex gloves	Fisher Scientific	19-014-643
General purpose face mask	Cardinal Health	AT7511
Bonnets	Medline	CRI1001
Shoe covers	U-line	S-7873W
Media and Reagents
Hanks’ Balanced Salt Solution (HBSS)	Life Technologies	14175095	without calcium or magnesium
TrypZean (animal product–free recombinant trypsin)	Sigma Aldrich	T3449
Soybean Trypsin Inhibitor  1g/50mL	Sigma Aldrich	T6522
Cryostor CS5	BioLife Solutions	205102
Trypan blue reagent	Life Technologies	15250-061
Anti-EpCam-PE	Miltenyi Biotec	130 - 091-253
PE-isotype control	Miltenyi Biotec	130-098-845
Anti-CD90-PeCy5	BD Pharmingen	555597
PeCy5-isotype control	BD Pharmingen	557224
Anti-CD105-APC	BD Pharmingen	562408
APC-isotype control	BD Pharmingen	340754
Collagen Type VI	Sigma Aldrich	C7521
Consumables
50 ml graduated pipette	BD/Falcon	356550
10 ml graduated pipette	BD/Falcon	356551
5 ml graduated pipette	BD/Falcon	356543
50 ml falcon tubes	BD/Falcon	352070
15 ml falcon tubes	BD/Falcon	352096
15 cm Petri dishes	Corning	351058
70 μm filters	BD/Falcon	352350
0.22 μm filters	Millipore	SLGV033RS
1 ml Pipette tips	Fisherbrand	02-707-401
200 μl Pipette tips	Fisherbrand	02-707-409
20 ml Syringe	BD/Medical	309661
Plastic spatula	Fisher Scientific	14-245-97
Plastic weighing boat	Fisher Scientific	02-202-102
Cryo vials	Nunc	377267
Equipment
Mr Frosty	Fisher Scientific	A451-4
Biohazard Cabinet