# Epicel^^ (cultured epidermal autografts)

## Background

Although the skin is often not thought of as an organ, it is the largest
organ system in the body and its specific and vital functions clearly
define it as such[^1]. These include:

-   Protective

-   Immunologic

-   Fluid, protein, and electrolyte homeostasis

-   Thermoregulation

-   Neurosensory

-   Metabolism

Skin is comprised of an epithelium (the epidermis), and a connective
tissue matrix (the dermis). The skin's primary function is to act as a
protective barrier between the body and the external environment. The
skin blocks penetration of microorganisms, absorbs and blocks radiation,
and inhibits the loss of water from the body. The skin also contains
several types of sensory receptors (pain, touch, hot and cold), is
involved in thermoregulation (prevents heat loss and allows for rapid
cooling through evaporation of sweat), and functions in immunologic
surveillance by assisting in the presentation of antigens to immune
cells.

The epidermis forms the outermost portion of the skin and is
approximately 0.04 to 1.5 mm in thickness compared with the 1.5 to 4 mm
of full thickness skin. It is a continuously regenerating, multilayered
(stratified), squamous epithelium that keratinizes. The epidermis is
composed of primarily epithelial cells or keratinocytes, as well as
other cell types, including melanocytes (pigment producing cells),
Langerhans cells and Merkel cells which serve as mechanoreceptors. The
epidermis is organized into several layers named for either a structural
property or a functional activity of the included cells.

The basal layer of keratinocytes contains young cells that are
mitotically active, providing generation of outwardly migrating
epidermal cell layers. From the basal layer, cells move outward and
mature in a process called differentiation. As keratinocytes
differentiate, mitosis no longer occurs and protein synthesis,
predominantly of keratins, becomes the dominant process. As the cells
move outward, they lose their nuclei, flatten and evolve into a dead
layer called the stratum corneum. This layer, is a compact, relatively
impervious layer that eventually desquamates. The stratum corneum layer
provides the majority of the skin's barrier function.

The dermis is a relatively thick layer comprised mainly of fibrous
connective tissue. The primary cell type is the fibroblast, a spindle
shaped cell which does not frequently replicate but is very active in
producing extracelluar proteins, primarily collagen and elastin. The
dermis makes up the bulk of the skin and provides its structural
strength. The dermis protects the body from mechanical injury, binds
water to serve as a water storage organ; it functions in thermal
regulation and as a receptor of sensory stimuli.

## Burn Injuries

Burn injuries can compromise all of these essential functions and are
classified in terms of degrees or depth of injury, whether or not they
involve additional trauma or smoke inhalation. First degree burns and
superficial second degree burns involve only the epidermal layer and,
perhaps, superficial portions of the dermal layer. Healing will
generally occur rapidly and completely within one to two weeks through
migration to the surface of epithelial cells, which survive in deeper
portions.

In deep partial thickness and full thickness burns (second and third
degree burns), most or all of the dermis is destroyed. Due to the depth
from which migrating epithelial cells must travel, reepithelialization
is greatly retarded or impossible. These wounds are routinely treated
with excision and grafting.

Fourth degree injury refers to situations where heat damage extends to
deep structures, such as muscle, tendon, bone, etc. Treatment may
require elaborate debridement or even amputation.

## Complications of the Burn Injury

The burn wound may be complicated by additional trauma sustained in the
course of the accident (e.g., fractures, lacerations from falls or lung
damage from smoke inhalation). Also an individual's medical history will
impact survival and morbidity. Ryan et. al. reports smoke inhalation
along with burn wound injury increases a patient's risk factors for
mortality[^2]^,^[^3].

Permanent wound coverage must be achieved in a timely fashion to avoid
the many complications of the burn wound injury. Large open wounds cause
fluid level imbalances, increased susceptibility to infection and
reduced immune response. Lack of wound coverage and its sequelae can
create an environment of sepsis, which can lead to multi-system organ
failure and death.[^4]

## Existing Treatment Options {#existing-treatment-options .unnumbered}

Generally a patient is admitted to a specialized burn treatment center
if they meet criteria which has been established by the American Burn
Association[^5]. The criteria include:

-   Second or third degree burns of 10% total body surface area (TBSA)
    in children \<10 years old or adults \>50 years old;

-   Second degree burns of ≥ 20% TBSA at any age;

-   Third degree burns ≥10% TBSA at any age;

-   chemical burns that threaten functional or cosmetic impairment of
    face, hands, feet, genitalia, or perineum;

-   Burns with inhalation injury.

Once the patient is admitted to a burn institution, an accurate estimate
of the total body surface area burned is calculated and fluid
resuscitation measures begin. The wounds are treated with topical
antimicrobial therapy, which in most cases is silver sulfadiazene cream.
The depth of the injury will determine if the wound will heal on its own
with conservative measures (i.e. daily dressing changes) or whether
surgical intervention is required.

The conventional treatment for burn wound closure is excision of the
eschar or removal of the necrotic material and placement of a split
thickness skin graft (STSG). A STSG is harvested from an area of the
individual's unburned skin. It includes the epidermal layer, which
regenerates and a very thin portion of the dermal layer, which does not
regenerate. This donor skin is placed upon the freshly excised burn
wound and closure ensues. This may occur fairly rapidly in patients with
small TBSA burns where there is abundant unburned skin to harvest. The
donor site may be used again after it heals which can range from 6 - 10
days, depending on the thickness of the harvested graft.

In burn injuries, sparing autograft dermis is a therapeutic goal.
Harvested with conventional methods, autograft has a total thickness of
approximately 0.010-0.016 inches and contains a significant amount of
dermis with the epidermal layer. This is in contrast to thin epidermal
autograft which is approximately one half that of conventional autograft
and consists of the epidermis and a very small amount of dermis. The
total number of harvests of conventional autografts, which can be
obtained from unburned parts of a patient's body, is limited to
approximately one to three procedures. Limited availability of
autograft, especially in large burns, can be managed by thin autograft
harvesting techniques, which reduce the amount of dermal tissue
harvested with the autograft.

In a larger TBSA burn, usually \>30%, the donor skin must be meshed and
expanded in an attempt to cover a larger surface area with a smaller
amount of available skin. If the patient does not have enough donor skin
to cover the wound, the physician may need to consider alternative means
of permanent wound closure in order to save the patient' s
life[^6]^,^[^7].

The epidermal barrier, which is essential for life, is a central
mechanism in preventing dehydration and microbial infection. Traumatic
injuries, such as burns, can result in the permanent loss of epidermal
tissue through destruction of the proliferative (basal) keratinocyte
layer.

## Epicel (cultured epidermal autografts)

Epicel is manufactured from epidermal cells harvested from uninjured
skin, cultivated *in vitro* to create individual grafts which are then
supported by a moisture-retaining petrolatum impregnated gauze
dressing[^8]. The grafts are thin, diaphanous sheets of epidermal cells
that, when transplanted onto the wound bed, will lead to the
reconstitution of a functional epidermal layer.

Epicel is for use in patients who have sustained a deep dermal or full
thickness burn covering 30% or more of their total body surface area.
Epicel provides a means to close full-thickness wounds when the injured
area is so large that the number of potential donor sites is limited.
Full-thickness injuries are, those which completely destroy the
regenerative capacity of the epithelium. Re-establishment of the
epithelial layer can only occur by harvesting partial-thickness skin
grafts from autologous donor sites. In those cases in which the patient
does not have enough skin available to close their wounds, Epicel is the
only treatment option. No other device is available which can provide
these patients with a permanent autologous epidermal replacement.

During Epicel cultivation, the proliferative cells within the epidermis
are isolated from a full thickness skin biopsy and rapidly expanded
using the superior growth conditions of tissue culture such that
sufficient grafts can be generated to cover the patient's wounds. The
Epicel culture process utilizes routine mammalian cell culture
techniques including an irradiated murine 3T3 cell feeder layer that
supports early growth of epidermal keratinocytes. As the keratinocytes
proliferate on the culture flask, the feeder layer is displaced from the
flask and removed during subsequent culture media changes. A description
of the characterization testing conducted on the 3T3 cells used in the
Epicel™ manufacturing process can be found on page 9 of this document.

During Epicel graft cultivation the cells that comprise the nascent
sheet tightly join as they approach confluence forming a
protease-resistant cohesive unit. At the same time, proliferating cells
in direct contact with the tissue culture substrate initiate
stratification creating a multilayered sheet. The cell-cell contacts
that bind the sheet together are a fundamental property of
keratinocytes[^9]. Epicel grafts are enzymatically released as intact
sheets from the tissue culture substrate and attached to a petrolatum
impregnated gauze dressing in such a way that the basal layer of cells
will come into direct contact with the wound surface. The petrolatum
gauze dressing provides structural support and prevents desiccation
during transport of the grafts to the surgeon and through the initial
phases of engraftment to the wound and epithelial maturation.
Engraftment of Epicel grafts leads to a reconstitution of the epidermal
layer[^10].

The primary benefit of the Epicel product is that the total number of
grafts required to treat a patient can be produced from a single biopsy
of unburned skin. Patients suffering burns over a significant body
surface area can be completely covered regardless of the amount of
unburned skin available for split thickness skin grafts. This minimizes
the time to wound closure and minimizes the time in which the patient is
most susceptible to serious and potentially life threatening
complications. For example, Munster et al[^11] reported on a series of
patients treated with cultured epidermal autografts who had a
significantly reduced mortality rate (14%) when compared with control
patients (48%).

# The Importance of Epicel

Epicel (cultured epidermal autografts) is indicated for use in a
subgroup that represents the most severely injured patients in the burn
population. Approximately 2700 patients present to burn centers each
year with burns ≥30% of the total body surface area. Treatment for these
patients requires that the wounds be closed as rapidly as possible. The
Epicel product is one of the medical treatments in the surgeon's
armamentarium that allows for the treatment and closure of these wounds.
In those cases in which the patient does not have enough skin available
to close their wounds, Epicel is the only treatment option. No other
device is available which can provide these patients with a permanent
autologous epidermal replacement.

Although the incidence of these extensive burns is low, the production
of an autologous epidermal replacement requires an extensive investment
by the manufacturer. The episodic nature of these injuries necessitates
a well-maintained facility and staff that is ready to respond when
required.

Genzyme Tissue Repair maintains a state of the art cell culturing
facility that can respond to those fluctuations and consistently produce
a quality product. From the time of biopsy receipt, to the product
release, the product is managed by an extensive set of standard
operating procedures and well-trained personnel. These technicians are
knowledgeable in the in-process, final product, and environmental
controls needed to ensure that the Epicel product to be grafted on the
patient is clinically acceptable.

Products such as Epicel provide a clear benefit to a critically ill
population of patients who might otherwise not survive with conventional
treatment. Because the patient population is very small, FDA granted
Humanitarian Use Device designation for Epicel. HUD designation also
requires identification of the product cost and a demonstration that the
amount charged does not exceed the cost of research, development,
fabrication and distribution of the product. There is a very high cost
of developing and maintaining a state of the art program such as Epicel.
The additional requirements imposed by regulation of Epicel as a
xenotransplant product in accordance with the expanded definition, would
increase the burden to the patient, the burn center and the manufacturer
and delay the time to treatment without necessarily increasing the
benefit to the patient.

# Characterization of Swiss Mouse Embryo 3T3 Cell Bank used in Epicel Autograft Cultivation

Testing of the 3T3 Master cell bank and Master Working cell bank were
carried out by Microbiological Associates Inc. (now Bioreliance)
following the requirements in FDA's Points to Consider in the
characterization of a cell substrate for production of a biological
material.

## Master Cell Bank Testing

### [Isoenzyme Analysis]{.underline}

The identity of a species of origin of a cell line can be determined on
the basis of the electrophoretic mobilities and banding patterns of the
isoenzymes of a number of intracellular enzymes including lactate
dehydrogenase (LD), glucose-6-phosphate dehydrogenase (G6PD), malate
dehydrogenase (MD), nucleoside phosphorylase (NP), and peptidase B
(PepB). Cells are burst and the proteins recovered run on an
electrophoresis gel to determine the banding pattern. The migration
distances for each isoenzyme band are determined and compared to a
reference table with standard migration distances for different species.
The results obtained on the cells from the Master cell bank were
consistent with the expected species of origin for the test article
(i.e., mouse).

### [Bacterial and Fungal Contaminants]{.underline}

The direct inoculation method used for this testing meets or exceeds the
USP 23 and/or 21 CFR 610.12 requirements for sterility testing. Cells
are lysed and lysate inoculated into fluid thioglycollate broth and
soybean-casein digest broth (as required by the sterility test, 21CFR
610.12), pre-reduced peptone yeast glucose (PYG) medium (to detect
anaerobic contaminants), and Sabouraud-dextrose agar (to detect fungal
contaminants). The test article showed no evidence of fungal or
bacterial contamination in any of the culture conditions during the
observation period.

### [Mycoplasma]{.underline}

Mycoplasma testing was performed using a large sample volume and exceeds
recommendations in *the Points to Consider in the Characterization of
Cell Lines Used to Produce Biologicals* (CBER 1993). This assay is
designed to detect the presence of agar-cultivable and non-cultivable
mycoplasma species under aerobic and anaerobic growth conditions. For
agar-cultivable mycoplasma, a lysate of the test article was inoculated
onto Type A and Type B agar. For amplification of low level organisms,
lysate material was inoculated into broth culture followed by subculture
onto agar plates on days three, seven, and fourteen. Under these
conditions, the test article was negative for agar-cultivable
mycoplasma. For detection of non-cultivable mycoplasma, the test article
was inoculated directly onto cultures of Vero cells. After incubation
for three to five days, the cultures were stained with Hoechst
bisbenzimid stain and examined by ultraviolet microscopy. All wells of
Vero cells inoculated with the test article were negative for the
presence of mycoplasma.

### [*In Vitro* Assay for Viral Contaminants]{.underline}

The *in vitro* assay employs three indicator cell lines (MRC-5, human
diploid lung cells; Vero, African green monkey kidney cells; and
NIH/3T3, Swiss mouse embryo cells) to screen for potential viral
contaminants that may be present in the test article. The indicator
cells are inoculated with a low speed supernatant collected from a
thawed cell lysate, and observed at least three times per week for the
appearance of cytopathic effects (CPE). In addition, indicator cell
culture supernatants were tested for hemagglutination (HA) and
hemadsorption (HAD) using chicken, guinea pig, and rhesus monkey
erythrocytes at 4°C and 36°C. No cytopathic effects, hemadsorption, or
hemagglutination was observed in any of the indicator cell cultures
inoculated with the test article.

### [Test for the Presence of Inapparent Viruses]{.underline}

Since inapparent (latent) viruses may not always be detected by
inoculating a battery of indicator cells and observing cytopathic
effects or other indications, an *in vivo* screening assay was performed
on the test article. Aliquots of the test article were inoculated into
both adult and suckling mice, guinea pigs, and embryonated hens' eggs.
In all cases, multiple inoculation routes were employed to maximize
detection of infectious agents. Adult mice and guinea pigs were observed
for twenty eight days for clinical signs. Suckling mice were observed
for fourteen days, after which all surviving mice were euthanized using
cervical dislocation. A homogenate was prepared and inoculated into a
new group of suckling mice by the same routes and in the same volumes as
the original group. These newly inoculated mice were observed for a
period of fourteen days. Embryonated eggs were inoculated via two
routes, allantoic and yolk sac. Following allantoic inoculation, the
eggs were checked for viability at 1 and 3 days post-inoculation, and
fluid collected from the eggs was tested for hemagglutination at 4°C (±
2°C) and 25°C (± 2°C) using chicken, guinea pig and human type O
erythrocytes. Pooled allantoic fluids were passaged to a new group of
embryonated eggs and observed and tested in the same way. Following yolk
sac inoculation, eggs were checked for viability at 1 and 10 days
post-inoculation. At 10 days, pooled yolk sacs were passaged into a new
group of embryonated eggs and were checked for viability at 1, 2 and 10
days post-inoculation. All adult and suckling mice, and guinea pigs
inoculated with the test article appeared normal and healthy during the
observation period. All hens' eggs inoculated via the allantoic or yolk
sac routes were viable and fluids were negative for hemagglutination. No
evidence of viral contamination was observed due to the test article.

### [Presence of Murine Specific Adventitious Agents]{.underline}

The Mouse Antibody Production test determines whether any of sixteen
murine specific adventitious agents were present. Viral antibody free
mice were inoculated with the test article via multiple routes to
maximize the potential immune response in the animals. Plasma or serum
from one group of mice was collected within 10 days of inoculation and
tested for lactate dehydrogenase (LDH) activity; elevated levels of this
enzyme are indicative of the presence of lactic dehydrogenase virus
(LDV). This same group was subsequently inoculated with a known lethal
strain of Lymphocytic Choriomeningitis Virus (LCMV), and monitored for
morbidity and mortality for up to 3 weeks. Animals would be protected
from this lethal challenge only if antibody to this virus is present.
The other groups of mice were bled no sooner than 28 days
post-inoculation and their serum assayed by ELISA, Hemagglutination
Inhibition (HI) or Indirect Fluorescent Antibody (IFA) assays for the
presence of antibody to known murine viruses. All sera from animals
inoculated with the test article were negative for the presence of
antibody to Ectromelia, GDVII, Lymphocytic Choriomeningitis (LCM),
Hantaan Virus, Minute Virus of Mice (MVM), Mouse Adenovirus, Mouse
Hepatitis Virus (MHV), Pneumonia Virus of Mice (PVM), Polyoma, Reovirus
Type 3, Epizootic Diarrhea of Infant Mice (EDIM), Mouse Salivary Gland
Virus (Mouse Cytomegalovirus - CMV), K, Mouse Thymic Virus (MTV) and
Sendai viruses as determined by ELISA, IFA, or HI. All plasma from
animals tested for lactate dehydrogenase activity showed normal levels
except for LDV inoculated control animals, which showed elevated levels
of LDH activity. All animals challenged with LCM virus died within 10
days of challenge, indicating that they were not protected by antibody
to LCMV produced in response to the original test inoculum. Based on the
data obtained in the assays performed, the test was shown to be free of
all of the 16 murine viruses for which it was examined.

### [Presence of Bovine Viruses]{.underline}

An *in vitro* assay was performed to determine whether the cells were
contaminated with any of five bovine viruses. Indicator cells specific
for these viruses, bovine turbinate cells, were inoculated with cell
lysate prepared from the test article and cultured for a period of 14
days. During this time, all cultures were monitored for cytopathic
effect (CPE). Any cultures exhibiting CPE were harvested and fixed when
approximately 50% of the monolayer was involved. All remaining cultures
were harvested and fixed after the 14 day incubation. The cells were
then examined by fluorescent antibody staining techniques for the
following bovine specific viruses: Bovine Viral Diarrhea virus (BVD),
Bovine Adenovirus Type 3 (BAV3), Bovine Parvovirus (BPV), Infectious
Bovine Rhinotracheitis virus (IBR), and Bovine Parainfluenza Virus Type
3 (PI3). None of the cultures inoculated with the test article exhibited
CPE. Fixed cells did not exhibit fluorescence when tested with antisera
specific for the 5 viruses used in this analysis. Based on these data,
the test article was determined to be free of the 5 specific bovine
viruses for which it was examined.

### [Retrovirology: Presence of Xenotropic Murine Retrovirus]{.underline}

The extended S^+^L^-^ focus assay was used to test the cells for the
presence of infectious xenotropic retrovirus. The development of foci in
the mink S^+^L^-^ cells is indicative of the presence of xenotropic
retrovirus contamination. The test article was inoculated directly onto
mink S^+^L^-^ cells as well as onto mink lung cells (extended portion)
for amplification of potential low level contaminants. After 2 passages
on the mink cells, culture fluids were harvested and assayed in the
S^+^L^-^ focus assay. No foci were observed on either the direct or
extended portions of this assay for cultures inoculated with the test
article. Based on these data, the test article was found to be negative
for xenotropic murine retrovirus.

### [Presence of Ecotropic Murine Retrovirus]{.underline}

The extended XC plaque assay was used to test the cells for the presence
of infectious murine ecotropic retrovirus (both N-tropic and B-tropic).
The test article was inoculated onto SC-1 cells for both the direct and
extended portions of the assay. The extended portion is used to amplify
potential low level contaminants. In the direct portion, the cultures
were incubated for 5-6 days, irradiated, and overlaid with XC cells. In
the extended portion, the cells were passaged twice, after which the
culture fluids were inoculated onto fresh SC-1 cells and processed as in
the direct assay. No plaques were observed in either the direct or
extended portions of the assay for cultures inoculated with the test
article. Based on these data, the test article was found to be negative
for murine retrovirus.

### [Presence of Viral-like Particles]{.underline}

Cells are examined by transmission electron microscopy (TEM) for the
presence of virus-like particles. Cells are pelleted and fixed with
Trump's fixative. Thin sections of the cell pellet are cut, mounted, and
stained with 5% methanolic uranyl acetate and Reynold's lead citrate.
The cell types are characterized via a morphological examination. Two
hundred test article cells are examined for any particles with a
virus-like morphology. The number of viral particles are tabulated and
the morphology of each particle examined to determine whether it is A-,
B-, C-, D-, and R-type retrovirus. Examination of the fixed test article
cells revealed no identifiable virus-like or retrovirus-like particles.

### [Presence of Retrovirus]{.underline}

A lysate prepared from the test article was assayed for
retroviral-derived reverse transcriptase activity as an indicator for
potential retrovirus contamination. The test article was incubated in
reaction mixtures containing the appropriate concentrations of Mg^++^ or
Mn^++^ to differentiate between C type and D type retrovirus reverse
transcriptase. All reaction mixtures containing the test article
(diluted or undiluted) incorporated less than 500 cpm of ^3^H-thymidine
above background. The diluted test article (1:2) did not inhibit
^3^H-thymidine incorporation in a control mixture containing Rauscher
Murine Leukemia Virus (R-MuLV). Based on these data, the test article
showed no evidence of the presence of type C or type D retrovirus
reverse transcriptase activity.

### [Tumorigenicity]{.underline}

The ability of the test article cells to form colonies in soft agar was
assessed as an indicator of possible transformation and the potential
ability for the cell line to form tumors in animals. A single-cell
suspension of test article cells were inoculated in a soft agar overlay
of a hard agar base. Cultures were monitored for 28 days for the
formation of colonies of greater than 10 cells. No colonies formed in
cultures inoculated with the test article cells.

## Master Working Cell Bank Testing

### [Bacterial and Fungal Contaminants]{.underline}

The direct inoculation method used for this testing meets or exceeds the
USP 23 and/or 21 CFR 610.12 requirements for sterility testing. Cells
are lysed and lysate inoculated into fluid thioglycollate broth and
soybean-casein digest broth (as required by the sterility test, 21CFR
610.12), pre-reduced peptone yeast glucose (PYG) medium (to detect
anaerobic contaminants), and Sabouraud-dextrose agar (to detect fungal
contaminants). The test article showed no evidence of fungal or
bacterial contamination in any of the culture conditions during the
observation period.

### [Mycoplasma]{.underline}

Mycoplasma testing was performed using a large sample volume and exceeds
recommendations in *the Points to Consider in the Characterization of
Cell Lines Used to Produce Biologicals* (CBER 1993). This assay is
designed to detect the presence of agar-cultivable and non-cultivable
mycoplasma species under aerobic and anaerobic growth conditions. For
agar-cultivable mycoplasma, a lysate of the test article was inoculated
onto Type A and Type B agar. For amplification of low level organisms,
lysate material was inoculated into broth culture followed by subculture
onto agar plates on days three, seven, and fourteen. Under these
conditions, the test article was negative for agar-cultivable
mycoplasma. For detection of non-cultivable mycoplasma, the test article
was inoculated directly onto cultures of Vero cells. After incubation
for three to five days, the cultures were stained with Hoechst
bisbenzimid stain and examined by ultraviolet microscopy. All wells of
Vero cells inoculated with the test article were negative for the
presence of mycoplasma.

### [*In Vitro* Assay for Viral Contaminants]{.underline}

The *in vitro* assay employs three indicator cell lines (MRC-5, human
diploid lung cells; Vero, African green monkey kidney cells; and
NIH/3T3, Swiss mouse embryo cells) to screen for potential viral
contaminants that may be present in the test article. The indicator
cells are inoculated with a low speed supernatant collected from a
thawed cell lysate, and observed at least three times per week for the
appearance of cytopathic effects (CPE). In addition, indicator cell
culture supernatants were tested for hemagglutination (HA) and
hemadsorption (HAD) using chicken, guinea pig, and rhesus monkey
erythrocytes at 4°C and 36°C. No cytopathic effects, hemadsorption, or
hemagglutination was observed in any of the indicator cell cultures
inoculated with the test article.

### [Test for the Presence of Inapparent Viruses]{.underline}

Since inapparent (latent) viruses may not always be detected by
inoculating a battery of indicator cells and observing cytopathic
effects or other indications, an *in vivo* screening assay was performed
on the test article. Aliquots of the test article were inoculated into
both adult and suckling mice, guinea pigs, and embryonated hens' eggs.
In all cases, multiple inoculation routes were employed to maximize
detection of infectious agents. Adult mice and guinea pigs were observed
for twenty eight days for clinical signs. Suckling mice were observed
for fourteen days, after which all surviving mice were euthanized using
cervical dislocation. A homogenate was prepared and inoculated into a
new group of suckling mice by the same routes and in the same volumes as
the original group. These newly inoculated mice were observed for a
period of fourteen days. Embryonated eggs were inoculated via two
routes, allantoic and yolk sac. Following allantoic inoculation, the
eggs were checked for viability at 1 and 3 days post-inoculation, and
fluid collected from the eggs was tested for hemagglutination at 4°C (±
2°C) and 25°C (± 2°C) using chicken, guinea pig and human type O
erythrocytes. Pooled allantoic fluids were passaged to a new group of
embryonated eggs and observed and tested in the same way. Following yolk
sac inoculation, eggs were checked for viability at 1 and 10 days
post-inoculation. At 10 days, pooled yolk sacs were passaged into a new
group of embryonated eggs and were checked for viability at 1, 2 and 10
days post-inoculation. All adult and suckling mice, and guinea pigs
inoculated with the test article appeared normal and healthy during the
observation period. All hens' eggs inoculated via the allantoic or yolk
sac routes were viable and fluids were negative for hemagglutination. No
evidence of viral contamination was observed due to the test article.

### [Tumorigenicity]{.underline}

The ability of the test article cells to form colonies in soft agar was
assessed as an indicator of possible transformation and the potential
ability for the cell line to form tumors in animals. A single-cell
suspension of test article cells were inoculated in a soft agar overlay
of a hard agar base. Cultures were monitored for 28 days for the
formation of colonies of greater than 10 cells. No colonies formed in
cultures inoculated with the test article cells.

## Production Cell Bank Testing

Production banks of 3T3 cells undergo raw material Quality Control
testing prior to acceptance for use in the Epicel manufacturing
process.

### [Source]{.underline}

Documentation accompanying each lot of Production 3T3 cells must specify
that these cells were grown from the 3T3 Master Working Cell bank.

### [Cell Number]{.underline}

Documentation accompanying each lot of Production 3T3 cells must specify
that there are [\>]{.underline} 1.4 x 10^8^ cells/vial.

### [Bacterial and Fungal Contaminants]{.underline}

Sterility testing is performed in-house by GTR's Quality Control
Department on 5% of each lot of Production 3T3s. The direct inoculation
method used for this testing is done according to USP "Sterility Tests"
\<71\>. Cells from each test vial are inoculated into and tryptic soy
broth to test for the presence of aerobic and fungal contaminants and
fluid thioglycollate medium to test for the presence of anaerobic
contaminants. The tryptic soy broth is incubated for fourteen days and
monitored for turbidity, which is an indicator of microbial growth.
Because the fluid thioglycollate medium has a gel base and is thicker
than tryptic soy broth, the inoculated 3T3s do not settle and the medium
remains cloudy. Therefore, in accordance with USP, between days 3 and 5,
1 mL of the medium is withdrawn and directly inoculated into another
bottle of fluid thioglycollate medium. This medium is then incubated for
fourteen days and monitored for turbidity. The test article must show no
evidence of fungal or bacterial contamination in any of the culture
conditions during the observation period to be acceptable for use.

### [Mycoplasma]{.underline}

Mycoplasma testing is performed using three vials of cells from each
Production cell bank. For each vial, five flasks of cells are plated and
grown in antibiotic-free medium. Cells are scraped from the flasks and
the cells and supernatants pooled. Two aliquots from the pooled sample
are sent to Bionique Testing Laboratory for mycoplasma testing in
accordance with the procedure outlined in *the Points to Consider in the
Characterization of Cell Lines Used to Produce Biologicals* (CBER 1993).
This assay is designed to detect the presence of agar-cultivable and
non-cultivable mycoplasma species under aerobic and anaerobic growth
conditions. All samples must be negative for mycoplasma in order to be
acceptable for use.

## *Irradiated* Production 3T3 Testing

Production 3T3 cells were tested for retroviral activation, as a result
of the cells receiving a radiation dose of 6000 rads during the
manufacturing process. An amp of Production 3T3 cells was thawed and
irradiated in accordance with current manufacturing practices. The
irradiated cells (either frozen or in monolayer culture) were sent to
Bioreliance to undergo various retroviral tests.

### [Retrovirology: Presence of Xenotropic Murine Retrovirus]{.underline}

The extended S^+^L^-^ focus assay was used to test the cells for the
presence of infectious xenotropic retrovirus. The development of foci in
the mink S^+^L^-^ cells is indicative of the presence of xenotropic
retrovirus contamination. The test article was inoculated directly onto
mink S^+^L^-^ cells as well as onto mink lung cells (extended portion)
for amplification of potential low level contaminants. After 2 passages
on the mink cells, culture fluids were harvested and assayed in the
S^+^L^-^ focus assay. No foci were observed on either the direct or
extended portions of this assay for cultures inoculated with the test
article. Based on these data, the test article was found to be negative
for xenotropic murine retrovirus.

### [Presence of Ecotropic Murine Retrovirus]{.underline}

The extended XC plaque assay was used to test the cells for the presence
of infectious murine ecotropic retrovirus (both N-tropic and B-tropic).
The test article was inoculated onto SC-1 cells for both the direct and
extended portions of the assay. The extended portion is used to amplify
potential low level contaminants. In the direct portion, the cultures
were incubated for 5-6 days, irradiated, and overlaid with XC cells. In
the extended portion, the cells were passaged twice, after which the
culture fluids were inoculated onto fresh SC-1 cells and processed as in
the direct assay. No plaques were observed in either the direct or
extended portions of the assay for cultures inoculated with the test
article. Based on these data, the test article was found to be negative
for murine retrovirus.

### [Presence of Viral-like Particles]{.underline}

Cells are examined by transmission electron microscopy (TEM) for the
presence of virus-like particles. Cells are pelleted and fixed with
Trump's fixative. Thin sections of the cell pellet are cut, mounted, and
stained with 5% methanolic uranyl acetate and Reynold's lead citrate.
The cell types are characterized via a morphological examination. Two
hundred test article cells are examined for any particles with a
virus-like morphology. The number of viral particles are tabulated and
the morphology of each particle examined to determine whether it is A-,
B-, C-, D-, and R-type retrovirus. Examination of the fixed test article
cells revealed no identifiable virus-like or retrovirus-like particles.

### [Presence of Retrovirus]{.underline}

A lysate prepared from the test article was assayed for
retroviral-derived reverse transcriptase activity as an indicator for
potential retrovirus contamination. The test article was incubated in
reaction mixtures containing the appropriate concentrations of Mg^++^ or
Mn^++^ in order to differentiate between C type and D type retrovirus
reverse transcriptase. All reaction mixtures containing the test article
(diluted or undiluted) incorporated less than 500 cpm of ^3^H-thymidine
above background. The diluted test article (1:2) did not inhibit
^3^H-thymidine incorporation in a control mixture containing Rauscher
Murine Leukemia Virus (R-MuLV). Based on these data, the test article
showed no evidence of the presence of type C or type D retrovirus
reverse transcriptase activity.

## End-Of-Production 3T3 Testing

Testing was conducted on production 3T3s which had been grown several
population doublings beyond the point at which they would be used in the
Epicel process. An amp of Production 3T3 cells was thawed in accordance
with the current manufacturing process and plated into culture using the
medium which is used by Quality Control to grow 3T3 cells for raw
material testing. The cells were sent to Bioreliance (tumorigenicity)
and Applied Genetics Laboratory (karyology) for testing.

### [Tumorigenicity]{.underline}

The ability of the test article cells to form colonies in soft agar was
assessed as an indicator of possible transformation and the potential
ability for the cell line to form tumors in animals. A single-cell
suspension of test article cells were inoculated in a soft agar overlay
of a hard agar base. Cultures were monitored for 28 days for the
formation of colonies of greater than 10 cells. The cultures were
equivalent to that acceptable for negative control.

### [Karyology]{.underline}

The cells underwent karyological analysis to determine the number of
chromosomes and to check for the presence of abnormalities. Cells are
swollen, fixed onto slides, and stained with Giemsa stain. One hundred
cells in metaphase were examined for chromosome count. Fifty of these
metaphases were examined for cytogenetic aberrations. The types of
chromosomal aberrations present were classified. Two chromosome
aberrations were found with 2% cells aberrant. Cytogenetic analysis
showed that the cells were of mouse origin. There was a wide variety of
normal chromosomes and chromosomal rearrangements, as is consistent with
a cell line. The modal chromosome number was 60 (range 47 to 128) and
chromosomes 18 and Y were absent in all karyotypes. There results are
consistent with the results of karyology on a sample of this cell line
deposited with ATCC, which had a modal number of 68, no Y chromosomes,
and very few cells with chromosome 18.

## Characterization of Epicel Final Product

Characterization testing was conducted on three lots of Epicel final
product. Because the final product was an intact sheet of cells which
could not be disaggregated without damaging the cells, testing was
performed on keratinocytes in culture prior to achieving confluence
(formation of an intact sheet). Lots of Epicel were grown by
Manufacturing in accordance with current practices. The 3T3s were
removed from the culture flasks by washing and shaking with EDTA. The
culture flasks with only keratinocyte colonies remaining were sent to
Bioreliance (tumorigenicity) and Applied Genetics Laboratory of Genzyme
Genetics (karyology) for testing.

### [Tumorigenicity]{.underline}

The ability of the test article cells to form colonies in soft agar was
assessed as an indicator of possible transformation and the potential
ability for the cell line to form tumors in animals. Typically, a
single-cell suspension of test article cells is inoculated in a soft
agar overlay of a hard agar base. Because the sample consisted of
keratinocyte colonies which did not easily disaggregate into single
cells, the protocol was modified to include a baseline colony count of
the agar plates at 24 hours. All cells in the entire plate were
examined. If any aggregates greater than 10 cells were present at 24
hours, these were attributed to incomplete disaggregation, and the test
was discontinued. Cultures were monitored for 28 days for the formation
of colonies of greater than the original baseline. No colonies formed in
cultures inoculated with the three lots of test article cells.

### [Karyology]{.underline}

Karyological analysis was conducted on cells to determine the number of
chromosomes and check for the presence of abnormalities. Cells are
swollen, fixed onto slides, and stained with Giemsa stain. Twenty
metaphases were examined for chromosome count and three metaphases were
karyotyped. In the one lot analyzed by Genzyme Genetics, the results
showed a normal 46,XX karyotype consistent with the patient's known sex.
There was no evidence of clinically significant numerical or structural
chromosome abnormalities, although this method would not detect small
rearrangements, microdeletions, and low level mosaicism. Karyotyping
studies on the last two lots are on-going at this time at Applied
Genetics Laboratory.

# 

[^1]: Herndon DN. Total Burn Care, 1996 Saunders Publ. Pg 35-36

[^2]: Ryan C, Schoenfeld D, Thorpe W. Objective Estimates of the
    Probability of Death From Burn Injuries. NEJM February 1998; 362-366

[^3]: Parks S. Inhalation Injury in Burn Patients. Trauma Rounds. West
    J. Med. 1976; 124:244-248

[^4]: Herndon DN. Total Burn Care. 1996 Saunders Publ. Pg 95-96

[^5]: Collini FJ, Kealy GP. Burns: A Review and Update. Contemp Surg.
    1989; 34:75-77

[^6]: McAree KG, Klein RL, Boeckman CR. The Use of Cultured Epithelial
    Autogratfs in the Wound Care of Severely Burned Patients. J. Pediatr
    Surg. 1993; 28:166-168

[^7]: Munster A. Cultured Skin for Massive Burns. Annals of Surg. 1997;
    Vol 224 No. 3 372-377

[^8]: Green H, Kehinde O, Thomas J (1979) Growth of cultured human
    epidermal cells into multiple epithelia suitable for grafting. PNAS
    USA 76(11): 5665-5668.

[^9]: Leigh IM and Watt FM (1994) The culture of human epidermal
    keratinocytes. In: The Keratinocyte Handbook, Chapter 2, Leigh, I,
    Lane B, and Watt F, eds, Cambridge University Press, pp 43-51.

[^10]: O'Conner NE, Muliken JB, Banks-Schlegel S, Kehinde O, and Green H
    (1981) Grafting of burns with cultured epithelium prepared from
    autologous epidermal cells. Lancet I: 191-199.

[^11]: Munster AM. Cultured Skin for Massive Burns A Prospective,
    Controlled Trial. Annals of Surgery 1996; 224:372-377