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.
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.
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 medicine1,2. 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 material. The properties that make hAECs appealing as a cellular therapy include their multipotency, low immunogenicity, and anti-inflammatory properties. hAECs have been found to be highly multipotent both in vitro and in vivo, capable of differentiating into mesodermal lineages (cardiomyocytes, myocytes, osteocytes, adipocytes), endodermal lineages (pancreatic cells, hepatic cells, lung cells) and ectodermal lineages (hair, skin, neural cells and astrocytes)5-10.
Reassuringly, despite their multipotency hAECs do not appear to either form tumors or promote tumour development in vivo. Furthermore, hAECs are also immune privileged, expressing low levels of class II human leukocyte antigens (HLAs)8. This property likely underlies their ability to evade immune rejection after allogeneic and xenogenic transplantation, as demonstrated in studies using immune competent monkeys, rabbits, guinea pigs, rats, and pigs11-13. hAECs display potent immunomodulatory and immunosuppressive properties and thus offer significant practical advantages for potential clinical applications in autoimmune disease therapy. hAECs are believed to exert immunomodulatory functions on both the innate and adaptive immune systems. One of the mechanisms suggested, is through the secretion of immunomodulatory factors14.
Current applications of hAECs in pre-clinical animal disease models include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Researchers have shown interest in using hAECs to treat post-stroke brain inflammation due to their unique properties. There is evidence that hAECs can cross the blood brain barrier where they can engraft, survive for up to 60 days, differentiate into neurons, decrease inflammation and promote regeneration of damaged central nervous system tissue in animal models of neurological diseases15.
hAECs offer the ability to target and reverse multiple pathological pathways that contribute to the development and progression of multiple sclerosis. For example, results from pre-clinical animal studies suggest that hAECs are strongly immunosuppressive and can potentially induce peripheral immune tolerance and reverse ongoing inflammatory responses. hAECs have also been shown to have the capacity to differentiate into neural cells in vivo and enhance endogenous neuroregeneration through the secretion of a vast array of neurotrophic factors16.
Human and rodent amnion epithelial cells have already demonstrated their therapeutic efficacy for the treatment of liver disease in animal models. In a carbon tetrachloride damage induction model of liver disease, hAEC transplantation lead to engraftment of viable hAECs in the liver, accompanied with reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis17.
hAECs can be stimulated to expressed pancreatic factors including insulin and glucose transporters. Several studies have investigated the potential for hAECs to restore blood glucose levels in diabetic mice18. In mice receiving hAECs, both animal body weight and blood glucose levels decreased to normal levels following injection of cells. These studies present a strong case for the use of hAECs for the treatment of diabetes mellitus.
hAECs have a proven role in the prevention and repair of experimental acute and chronic lung injury in both adult and neonatal models19. These studies found that hAECs differentiate in vitro into functional lung epithelial cells expressing multiple lung-associated proteins, including Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the ion channel that is mutated in patients with cystic fibrosis20. Additionally, when hAECs are delivered to the injured adult and neonatal lung, they exert their reparative effects via the modulation of host immune cells, reducing pulmonary leucocyte recruitment, including neutrophils, macrophages and lymphocytes21-23.
Given their abundance, safety record, and proven clinical applications for multiple diseases, clinical trials using hAECs is inevitable. With the goal of accelerating the translation of hAEC therapies into clinical-trials, we developed methods to isolate, cryopreserve and culture hAECs in a manner suitable for clinical trials, using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.
We based this protocol a previously published protocol that we were using successfully to isolate hAECs using animal-derived reagents6. We altered the original protocol to replace animal-derived products with animal product-free reagents, and subsequent optimization was performed to optimize cell yield, viability and purity. Our goal was to develop a protocol that would comply with regulatory standards for cell manufacturing for human clinical trials.
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
2. Cryopreservation of hAECs
3. Thawing and Culture of Cyropreserved hAECs
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 mesenchymal marker positive cells. These markers were selected due to their specificity for epithelial24,25 and mesenchymal26 lineages respectively. This process can take approximately 4-5 hr to complete (Figure 1).
Cryopreservation requires 20-60 min depending on the number of vials and final cell yield. We have previously tested a range of cryopreservation media and found that many animal product-free media perform suitably, however the optimal DMSO concentration is approximately 5-10%. Typically one can expect the viability post-thaw to be 5-10% less than pre-cryopreservation viability, and this loss can be significantly greater for the inexperienced user (Figure 2).
Cell culture can be performed with hAECs to enable characterization, differentiation, or other specific in vitro procedures. These cells initially attach to cell surfaces with an efficiency of 50-80% (personal observation). This increases to 70-90% with subsequent passages. Attachment may be increased with the use of surface coating materials such as collagen type I, or similar products. hAECs maintain their epithelial phenotype in serum-free culture media (Figure 3). We have found significant changes in cell surface marker profiles following repeated passage. Additionally, after approximately 5 passages hAECs can either reach senescence, or go through morphological changes consistent with epithelial to mesenchymal transition. After repeated passage, hAECs maintain a normal karyotype, long telomere lengths and cell cycle distribution. These properties indicate a low risk of transformation or tumorigenicity. In addition to possessing immune regulatory and anti-inflammatory properties, hAECs have been shown to be highly multipotent in vitro and in vivo, differentiating into cell lineages representative of the three primary germ layers (Figure 4).
Figure 1: Expected cell yield, viability and purity for clinical isolation protocol. Similar cell yields and viability can be achieved using animal-product free reagents compared to standard animal product-containing methods. A typical isolation should be >90% EpCAM and <1% CD90/CD105 positive cells. Please click here to view a larger version of this figure.
Figure 2: Post thaw viability and metabolism for serum-containing and serum-free cryopreservation media. Compared to FBS containing 10% DMSO, commercially available animal product-free cryopreservation media showed similar or increased post-thaw viability and metabolism. Please click here to view a larger version of this figure.
Figure 3: Typical attachment and growth profile of hAECs in serum-free and serum-containing cell culture medium. Animal product-free culture media showed suitable cell attachment, proliferation, and maintenance of epithelial phenotype. However further development of serum-free media is required to achieve equal results to serum-containing media. Scale bars represent 500 µm.
Figure 4: Multipotent differentiation of hAECs. At early passage, hAECs have been shown to differentiate into multiple cell lineages representative of the three primary germ layers. These lineages include, but are not limited to; neurons, hair and skin cells, lung epithelium, cardiomyocytes, hepatocytes, pancreatic cells, osteocytes and adipocytes. (Figure adapted from 27)
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 cell viability can decrease with extended storage time. We, and others have found variability in cell yield and viability, and to minimize this variability it is recommended that placenta are collected from healthy term births, preferably delivered by cesarean section.
We found that contamination of the amnion membrane with blood cells will result in inhibition of enzyme activity and a reduction in cell yield. Therefore a critical step in the protocol is thorough washing of the placenta and the amnion membrane is recommended. It is recommended that the amnion tissue be washed until it appears white/clear to avoid downstream enzyme inhibition. Isolation of a relatively homogeneous population of epithelial cells is important for characterization and quality control testing.
If low yields, viability or contamination of the cell yield with mesenchymal cells occurs there are several modifications that can be performed for troubleshooting. If cell yields are low, it is likely that enzymatic activity was inhibited due to blood or serum contamination. This can be resolved with more thorough and additional washing steps and/or discarding bloody or contaminated amnion pieces. Enzyme incubation time can also be increased, but this can be detrimental to cell viability. Decreased viability or contamination with mesenchymal cells can be avoided by decreasing the digest time. However, this may also decrease the total yield of cells. Times ranging from 30 min to 2 hr can be used for this protocol. These times need to be optimized for desired cell purity and total yield/viability. One limitation of this technique is that increasing cell yield or viability can come at the expense of each other. Additionally, serum-free components are currently not as effective of maintaining cell viability following exposure to enzymatic activity.
Cells can be cryopreserved without any major decrease in cell viability or loss of metabolism by storage in a temperature controlled liquid nitrogen system. This can range from a simple isopropanol-filled cryopreservation device to a state-of-the-art controlled rate freezing system. To our knowledge, the optimal freezing rate for hAECs in this system has not been determined, however the standard rate of 1 °C/min results in suitable maintenance of cell viability and post-thaw metabolism. During thawing of cryopreserved cells, is important to keep thawing time to a minimum to reduce cell exposure to DMSO. Cells may attach and proliferate at a lower rate following cryopreservation and thawing compared to culturing freshly isolated cells.
For clinical application it may be desirable to culture cells to increase cell number or for downstream characterization and/or in vitro manipulation. Readers should understand how their specific in vitro manipulation might alter the regulatory pathways involved in the clinical application of these cells. We investigated that an animal product–free culture medium such as EpiLife was suitable for the maintenance and expansion of hAECs. However, it is required to optimize the growth factor concentrations to achieve increased growth rates for such cell type. The issue of cell adherence during culture by using a human collagen-coating matrix, increases the plating efficiency. However, following prolonged exposure to enzymatic digestion the plating efficiency will be sub-optimal.
In summary, this protocol has been developed to facilitate the isolation and culture of human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. The advantage of this method compared to alternative isolation methods, 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.
The authors have nothing to disclose.
The authors acknowledge financial support from the Victorian Government’s Operational Infrastructure Support Program.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Collection Kit | |||
Stripping tray | Fisher Scientific | 13-361B | |
Liberty Dressing Forcep, pointed 13cm | Fisher Scientific | S17329 | |
Scissors – Sharp/Blunt Straight | Fisher Scientific | NC0562592 | |
Sterile latex gloves | Fisher Scientific | 19-014-643 | |
Protective Apparel (Gown) | U-line | S-15374-M | |
Protective Apparel | |||
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 | |||
50mL graduated pipette | BD/Falcon | 356550 | |
10mL graduated pipette | BD/Falcon | 356551 | |
5mL graduated pipette | BD/Falcon | 356543 | |
50mL falcon tubes | BD/Falcon | 352070 | |
15mL falcon tubes | BD/Falcon | 352096 | |
15-cm petri dishes | Corning | 351058 | |
70-μm filters | BD/Falcon | 352350 | |
0.22-μm filters | Millipore | SLGV033RS | |
1ml Pipette tips | Fisherbrand | 02-707-401 | |
200ul Pipette tips | Fisherbrand | 02-707-409 | |
20ml 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 |