KULTUR JARINGAN

Dr. Enni Suwarsi Rahayu,

M.Si
Dr.dr. Nugrahaningsih WH,
M.Kes

MATERI 9
BAHAN KULTUR
JARINGAN HEWAN

BAHAN KULTUR
JARINGAN HEWAN

Bahan

Media
sera
Reagents/cairan
Sel/jaringan

MEDIA
Merupakan bahan penting untuk kultur
Menyediakan nutrisi bagi sel dalam
kultur
Berdasar pada asalnya :
o Natural
o Buatan
Berdasar pada kadar serum yang
ditambahkan, ada 3 jenis media yaitu:
o basal media,
o reduced-serum media
o serum-free media

Types of Cell Culture Media

Natural
Artificial

MEDIA TYPE

Natural media

Artificial media

EXAMPLES

USES

plasma, serum,
lymph, human
Biological
placental cord
Fluids
serum,
amniotic fluid
Extract of liver,
spleen, tumors,
leucocytes and
bone marrow,
Tissue Extracts
extract of
bovine embryo
and chick
embryo
coagulants or
Clots
plasma clots
Balanced salt
solutions

PBS, DPBS,
HBSS, EBSS

Basal media

MEM DMEM
RPMI-1640,

Form the basis

of complex
media
Primary and
diploid culture
Supports wide
range of

Serum
Serum is vitally important as a
source of growth and adhesion
factors, hormones, lipids and
minerals for the culture of cells
in basal media.
Serum also regulates cell
membrane permeability and
serves as a carrier for lipids,
enzymes, micronutrients, and
trace elements into the cel

 Using serum in media has a number of

disadvantages including high cost,
problems with standardization,
specificity, variability, and unwanted
effects such as stimulation or inhibition
of growth and/or cellular function on
certain cell cultures.
If the serum is not obtained from
reputable source, contamination can also
pose a serious threat to successful cell
culture experiments.

Media Basal
media yang mengandung bahan
dasar untuk pertumbuhan
Mengandung : asam amino,
vitamin, garam anorganik,
glukosa
Harus ditambah serum

Reduced-serum Media
Merupakan strategi untuk
mengurangi efek tidak
diinginkan akibat adanya serum
Formula media ini diperkaya
dengan nutrien dan animalderived factors , yang berfungsi
mengurangi jumlah penggunaan
serum dalam media

Serum-Free Media
Menggantikan serum dengan
animal sera dengan tujuan untuk
memberikan nutrisi dan hormon
yang sesuai
Keuntungan utamanya: dapat
menjadi media selektif untuk
beberapa kultur primer

Keuntungan Serum-free
Media
Increased definition
More consistent performance
Easier purification and downstream
processing
Precise evaluation of cellular functions
Increased productivity
Better control over physiological
response
Enhanced detection of cellular
mediators

Kerugian Serum-free Media

Requirement for cell typespecific media formulations
Need for higher degree of
reagent purity
Slower growth

Basic Components of Culture Media

amino acids,
glucose,
salts,
vitamins,
hormone
other nutrients,

Amino Acids
Amino acids are the building blocks
of proteins
not synthesize these by themselves
required for the proliferation of cells
their concentration determines the
maximum achievable cell density
L-glutamine, an essential amino acid,
is particularly important

Carbohydrates
Carbohydrates in the form of sugars
are the major source of energy.
Most of the media contain glucose
and galactose,
however, some contain maltose and
fructose.

Vitamins
Many vitamins are essential for growth and
proliferation of cells.
Vitamins cannot be synthesized in sufficient
quantities by cells
important supplements required in tissue culture.
Again serum is the major source of vitamins in
cell culture, however, media are also enriched
with different vitamins making them suitable for
a particular cell line.
The B group vitamins are most commonly added
for growth stimulation.

Trace Elements
Trace elements are often supplemented to
serum-free media to replace those normally
found in serum.
Trace elements like copper, zinc, selenium
and tricarboxylic acid intermediates are
chemical elements that are needed in
minute amounts for proper cell growth
These micronutrients are essential for many
biological processes, e.g. the maintenance
of the functionality of enzymes.

Antibiotics
Although not required for cell growth,
antibiotics are often used to control the
growth of bacterial and fungal
contaminants.
Routine use of antibiotics for cell culture is
not recommended since antibiotics can
mask contamination by mycoplasma and
resistant bacteria .
Moreover, antibiotics can also interfere with
the metabolism of sensitive cells.

SERUM-CONTAINING MEDIUM (EAGLES MEDIUM)

Essential amino acids

The essential amino acidshistidine, isoleucine,

leucine, lysine, methionine, phenylalanine,
threonine, tryptophan, and valineplus cysteine,
glutamine, and tyrosine (all at 104 to 105 M)

Vitamins

Choline, folic acid, nicotinamide, pantothenate,

pyridoxal, and thiamine (all at 1 mg/L); inositol (2
mg/L); riboflavin (0.1 mg/L)

Salts

Na+, K+, Ca2+, Mg2+, Cl, PO43, HCO3

Glucose

0.9 g/L

Dialyzed serum*

510% of total volume

DEFINED (SERUM-FREE) MEDIUM

Amino acids

As above plus alanine and asparagine (104 M)

Vitamins, salts, glucose

As above

Other additions:
Fatty acids

Linoleic acid, lipoic acid

Nitrogen compounds

Hypoxanthine, thymidine, putrescine

Carbon source

Pyruvate and glucose (0.9 g/L)

Trace elements

Cadmium (Cd), manganese (Mn), molybdenum

(Mo), nickel (Ni), tin (Sn), vanadium (V)

Hormones and growth factors

Insulin, transferrin, hydrocortisone, fibroblast

growth factor, epidermal growth factor

SEL
Dari donor
Sel line

Isolasi Limfosit Dari Darah

Tepi
Sebelum
diberikan
perlakuan,
darah tepi
diambil
masing-masing
sebanyak 40
ml melalui
vena jugularis.

Darah ditampung dalam tabung yang

telah diisi antikoagulan EDTA 5%.
Dari darah tersebut diisolasi limfosit
dengan teknik picoll-paque gradient
(Sigma, USA).
Pengambilan darah dan isolasi limfosit
dengan cara yang sama dilakukan pada
bulan ke 3 dan 10.
Darah disentrifus 2500 rpm selama 10
menit.
Lapisan putih (buffy coat) di antara sel
darah merah dan plasma diambil dan
disuspensikan dengan media dulbeccos
modified eagles medium (DMEM) tanpa
serum.

Lanjutan...
Sel limfosit dipisahkan dari buffy coat
dengan cara Ficoll-paque gradient yaitu
disentrifus 3.000 rpm selama 30 menit.
Lapisan sel limfosit diambil dan dicuci tiga
kali dengan DMEM tanpa serum.
Limfosit yang diisolasi dilakukan uji
methylthiazol tetrazolium (MTT) untuk
mengetahui respons kekebalan selulernya.

Teknik Isolasi Limfosit dari Limpa

Alat dan bahan

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.

Alat

Sarung tangan disposable

Botol semprot
Alat bedah steril
Syringe 5 ml steril
Cawan petri steril
Botol steril
Wadah bertutup (toples),
Pipet 5 ml atau 10ml
disposable
Mikropipet 10-100l and tip
Tube 1,5 ml
Rak tabung sentrifus
Sentrifus
Object galss
Deck glass
Mikroskop

Bahan

1. Alkohol 70%
2. Kapas
3. Tissue
4. Aluminium foil
5. Kloroform
6. PBS steril
7. Garam fisiologis
8. Kertas saring
9. Methylene blue
10.Tikus /mencit

Cara kerja
Tikus jantan dibius dengan cara
dimasukkan ke dalam toples yang
berisi kapas yang telah diberi
kloroform

Setelah tikus pingsan, seluruh

badan tikus dibasuh dengan
alkohol 70%
Tikus diletakkan pada meja
bedah
Dibedah bagian perutnya untuk
diambil limpa secara steril
Limpa dimasukkan ke dalam botol
steril berisi 3 ml PBS steril.

Lanjutan
Limpa dipindahkan ke dalam cawan petri
steril berisi 5 ml PBS steril.
Limpa digerus menggunakan ujung
syringe kemudian disarimg menggunakan
kertas saring.
Suspensi sel dipindahkan ke dalam tube
1,5ml.

Disentrifus 1500 rpm selama 10 menit

Cairan supernatan dibuang, Pelet sel

dijentik-jentikkan agar pelet hancur (tidak
kompak)

Menambahkan pellet dengan PBS steril

secukupnya lalu membuat apusan
menggunakan object glass.

Melakukan pengecatan menggunakan

methylene blue selama 15 menit lalu
mencucinya dengan air mengalir.

Mengamati sel limfosit menggunakan

mikroskop dari perbesaran lemah hingga
kuat.

TERIMA KASIH
ATAS
PERHATIANNYA