HOW CELL GROW&Batch Growth Kinetics

8/20/2019 HOW CELL GROW&Batch Growth Kinetics

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Dhena RiaBarleany

UNTIRTA2015

HOW CELL

GROW


2/46

CLASSIFICATION OF CELL CULTURES


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MAING A !RIMAR" CULTURE


4/46

e#$i%&en'(GROWING CELLS IN

CULTURE


5/46

CO2 incubator

&ain'ain( CO2

le)el *5+10,-.h$&i/i'y an/

'e&%era'$re * C- ' (i&$la'e in)i) 3n/i'in(4


6/46

Water bath

T ar& &e/ia.6e7re %la3in8 n3ell(

Can har6r 7$n8i an/6a3'eria. (%ray alli'e&( i'h 0,e'hanl 6e7re

%la3in8 in 'he h/4 U($ally 'a9e( 10 +15

&in$'e( 7r &e/ia 'ar&. 5+10 7r TRED

' 'ha


7/46

Vacuum pump

Fr %er&anen'a(%ira'in 7li#$i/( *&e/ia.

!BS an/ TRED-4 U(e $n%l$88e/

8la(( %a('e$r

%i%e'(. 'hr in'(har%( 6: hen/ne4


8/46

Inverted PhaseMicroscope

A %ha(e 3n'ra('&i3r(3%e i'h6;e3'i)e( below 'he

(%e3i&en4 A %ha(e %la'e i'h an

ann$l$( ill ai/ ine:%li'in8 /i


9/46

Mechanics of phasemicroscopy

Shifting of phase by ½ a wavelength

Add and subtract amplitudes to create

more contrast


10/46

A comparison

Figure 1: The same cells imaged with

traditional bright field microscopy (left)

and with phase contrast microscopy

(right).


11/46

Hemacytometer

S%e3iali=e/3ha&6er i'he'3he/ 8ri/ $(e/

' 3$n' 'hen$&6er 7 3ell( ina (a&%le4

$(e 7 'ry%an 6l$eall(/i


12/46

Using theHemacytometer

Re&)e 'he he&a3y'&e'er

an/ 3)er(li% *3are7$lly-

7r& E'OH an/ /ry

'hr$8hly i'h a 9i&i%e4

Cen'er 3)er(li% n

he&a3y'&e'er

Barely >ll 'he 8ri/ $n/er 'he3)er(li% )ia 'he /i)e' i'h

y$r 3ell ($(%en(in4

C$n' 3ell( in 'en (#$are( *5

n ea3h (i/e- 6y 7llin8


13/46

oo!ing atthe grid

under thephase

contrastmicroscope


14/46

H 'he 3ell( ill a%%ear

Bri8h' re7ra3'ile ?(%here(@

are li)in8 3ell(.

Bl$e 3ell( a6$' 'he (a&e

(i=e a( 'he 'her 3ell( are/ea/4

ee% a /i


15/46

Count 10 squares

Any 10 will do but we

will follow convention

Watch for stringy, reddish

material—those aren’t cells!

serum


16/46

o grou

Count cells that

touch to and

left lines

"# $#Count cells thattouch bottom and

right lines


17/46

%ottom &rou


18/46

Ca"cu"ate your ce""s#m"

Cal3$la'e 'he n$&6er 7 ''al3ell( in ne &l 7 y$r($(%en(in4

$ota" ce""s counted % &di"ution factor' %

&()*)))'

number of s+uares

Here. /il$'in 7a3'r i( 2 an/ 7 (#$are( i( 10

*$r e:a&%le 210 :2 :10

142 : 105


19/46

,etermine your percentviabi"ity

ia6ili'y i( a &ea($re h &any 7y$r 3ell( ($r)i)e/ y$r 3ell 3$l'$re'e3hni#$e4

7 )ia6le *li)in8- 3ell( : 100

''al n$&6er 7 3ell( 3$n'e/

O$r e:a&%le 52 : 100 40J,


20/46

Aseptic $echni+ue

Fr 6e(' re($l'( in 'i(($e 3$l'$re. e an' ' r9' 9ee% &i3r6ial *6a3'eria. yea(' an/ &l/(-3n'a&ina'in ' a &ini&$&4 T / 'hi(. 'here

are 3er'ain 'hin8( y$ &$(' 6e aare 7 an/8$i/eline( ' 7ll4 Wr9 in a 3$l'$re h/ (e'+a(i/e 7r 'i(($e 3$l'$re

%$r%(e(4 M(' ha)e >l'ere/ air 'ha' 6l( a3r(('he ($r7a3e ' 9ee% &i3r6e( 7r& (e''lin8 in 'heh/4 T$rn < 'he Uan'i&i3r6ial li8h' an/ '$rnn 'he h/ 0 &in$'e( %rir ' en'erin8 'heh/4

Wear (hr' (lee)e( r rll y$r (lee)e( $%4 T$rny$r 6a(e6all 3a%( 6a39 i7 y$ MUST ear 'he&.'ie ln8 hair 6a39 an/ re&)e rin8( an/ a'3he(4


21/46

Wa(h han/( i'h (a% an/ a'er 6e7re6e8innin8 'he %r3e/$re an/ rea(h i7 y$'$3h any'hin8 'ha' i( n' ('erile r i'hin'he h/4

S%ray /n y$r han/(. r9 ($r7a3e. an/any'hin8 'ha' ill 8 in' 'he h/ i'h0, e'hanl4 Rei%e a' in'er)al( i7 y$are r9in8 7r a ln8 'i&e in 'he h/4

Thi( ill re/$3e 'he n$&6er( 7 6a3'eriaan/ &l/ 3n(i/era6ly4 D n' 6rea'he /ire3'ly in' y$r 3$l'$re(.

6''le( 7 &e/ia. e'34 Thi( al( &ean( '9ee% 'al9in8 ' a &ini&$&4 N (in8in8 r

3hein8 8$&4


22/46

Wr9 a( #$i39ly a( y$ 3an i'hin li&i'( 7 y$r3r/ina'in4 Al(. 9ee% 6''le( an/ Ka(9( 3l(e/hen y$ are n' r9in8 i'h 'he&4 A)i/%a((in8 y$r ar& r han/ )er an %en 6''le4

U(e nly ('erili=e/ %i%e'(. %la'e(. Ka(9( an/6''le( in 'he h/ 7r %r3e/$re(4

Ta9e (%e3ial %re3a$'in( i'h 'he ('erile %i%e'(4Re&)e 'he& 7r& 'he %a39a8e ;$(' 6e7re $(e4Ma9e 3er'ain ' (e' $% 'he n$&6er( n 'he %i%e'( 'ha' 'hey 7a3e y$4 Ne)er &$'h+%i%e'. $(e 'he%i%e''in8 ai/4 Chan8e %i%e'( 7r ea3h&ani%$la'in4 I7 'he 'i% 7 'he %i%e' '$3he((&e'hin8 $'(i/e 7 'he Ka(9 r 6''le. re%la3e

i'h a ne ne4 Ne)er $(e a %i%e' 'i3e4


23/46

Cell Gr'h ine'i3(

In'r/$3'in Gr'h %a''ern( an/ 9ine'i3( in 6a'3h

3$l'$re

+ 8r'h %ha(e(+ e


24/46

Gr'h ine'i3(

In'r/$3'in+ A$'3a'aly'i3 rea3'in The ra'e 7 8r'h i( /ire3'ly rela'e/ '

3ell 3n3en'ra'in

($6('ra'e( 3ell( e:'ra3ell$lar %r/$3'( &re 3ell(

S P ! nP

S ($6('ra'e 3n3en'ra'in *8L-Q P 3ell &a(( 3n3en'ra'in *8L-Q

! %r/$3' 3n3en'ra'in *8L-Q n in3rea(e/ n$&6er 7 6i&a((4

Ne' (%e3i>3 8r'h ra'e *1'i&e-

' 'he 'i&e

dt

dX

X

1net ≡ µ


25/46

Gr'h ine'i3(

In'r/$3'inNe' (%e3i>3 8r'h ra'e *1'i&e-

Gr(( (%e3i>3 8r'h ra'e *1'i&e-

The ra'e 7 l(( 7 3ell &a(( /$e ' 3ell /ea'hr en/8en$( &e'a6li(&

En/8en$( &e'a6li(& /$rin8 'he ('a'inary %ha(e.'he 3ell 3a'a6li=e( 3ell$lar re(er)e( 7r ne 6$il/in8 6l39( an/7r ener8y+%r/$3in8 &n&er(4

d k g −= µ µ net

: g µ

:d k


26/46

Gr'h ine'i3(

In'r/$3'inNe' (%e3i>3 re%li3a'in ra'e *1'i&e-

Gr(( (%e3i>3 re%li3a'in ra'e *1'i&e-

The ra'e 7 3ell /ea'h *1'i&e-

N Cell n$&6er 3n3en'ra'in *3ell n$&6er L-

dt

dN

N

1

R

≡ µ

:' R

µ

:d k

R µ

d k −= 'R R

µ µ


27/46

Gr'h ine'i3(

In'r/$3'in+ $an'i7yin8 3ell 3n3en'ra'in

+ /ire3' n ($(%en/e/ (li/ an/ in'er7eren3e3&%$n/(4

3ell &a(( 3n3en'ra'in %re7erre/

/ry ei8h'. %'i3al /en(i'y *'$r6i/i'y- *00+00n& Wa)eLen8'h-

3ell n$&6er /en(i'y

!e'r


28/46

Gr'h ine'i3(

+ $an'i7yin8 3ell 3n3en'ra'in

+ in/ire3' /ire3' &e'h/ i( ina%%li3a6le4 *&l/(li/ ('a'e 7er&en'a'in-

Cell &a(( 3an 6e /e'er&ine/ 6y&ea($re&en' 7 %r'ein. DNA r AT!4 e484 1&8AT!8 /ry ei8h' 6a3'erial 3ell4

I7 100 &8 AT!L i( &ea($re/. 'hen 'he 3ell &a((

100 &8 *AT!L-1 &8 AT!8 /ry 3ell(100 *8 /ryei8h' 3ell(L-


29/46

Gr'h ine'i3(

Gr'h %a''ern( an/ 9ine'i3( in 6a'3h3$l'$re

+ 8r'h %ha(e(

In 6a'3h 3$l'$re

+ la8 %ha(e

+ l8ri'h&i3 r e:%nen'ial 8r'h

%ha(e+ /e3elera'in %ha(e

+ ('a'inary %ha(e

+ /ea'h %ha(e


30/46

Ty%i3al 8r'h 3$r)e 7r a 6a3'erial %%$la'in


31/46

ag phase

A %eri/ 7 a/a%'a'in 7r 'he 3ell( ''heir ne en)irn&en'

Ne en=y&e( are (yn'he(i=e/4

A (li8h' in3rea(e in 3ell &a(( an/ )l$&e. 6$' nin3rea(e in 3ell n$&6er

!rln8e/ 6y l in3$l$& )l$&e. %r in3$l$&3n/i'in *hi8h , 7 /ea/ 3ell(-. a8e 7 in3$l$&. n$'rien'+%r &e/i$&

• M$l'i%le la8 %ha(e( */ia$:i3 8r'h- &e/i$& 3n'ain(&re 'han ne 3ar6n ($r3e

Ba'3h Gr'h ine'i3(


32/46


Dia$:i3 8r'h


33/46



34/46

Ba'3h Gr'h ine'i3(

-%ponentia" gro.th phase

In 'hi( %ha(e. 'he 3ell( ha)e a/;$('e/ ''heir ne en)irn&en' an/ &$l'i%lyra%i/ly *e:%nen'ially-

Balan3e/ 8r'h all 3&%nen'( 7 a 3ell8r a' 'he (a&e ra'e4

Gr'h ra'e i( in/e%en/en' 7 n$'rien'3n3en'ra'in. a( n$'rien'( are in e:3e((4


35/46

Ba'3h Gr'h ine'i3(


The 6alan3e 7 3ell &a(( in a 6a'3h 3$l'$re8i)e(

0tandtat timeionsconcentratcellare0XandX

0or,0

ln

:yieldsequationabovetheof nIntegratio

0 0 ,

=

==

===

t e X X t net μ

X

X

t at X X X net dt

dX

net µ

µ


36/46


constant.isslopehe net µ


37/46

Ba'3h Gr'h ine'i3(


D$6lin8 'i&e 7 3ell &a(( 'he 'i&e re#$ire/ ' /$6le 'he&i3r6ial &a((

!1"time#rategro$thspecificma%imumtheis

R

m μ

m μnet == µ µ

netnetnet

&(.0)ln0"ln

µ µ µ τ ===

X X d


38/46


39/46

Ba'3h Gr'h ine'i3(

,ece"eration gro.th phase

ery (hr' %ha(e. /$rin8 hi3h 8r'h

/e3elera'e( /$e ' ei'her

De%le'in 7 ne r &re e((en'ialn$'rien'(

The a33$&$la'in 7 ':i3 6y+%r/$3'( 78r'h *e484 E'hanl in yea('7er&en'a'in(-

!eri/ 7 $n6alan3e/ 8r'h Cell(


40/46



41/46

ine'i3 !a''ern 7 Gr 'h an/


42/46

ine'i3 !a''ern 7 Gr'h an/!r/$3' Fr&a'in

Gr'h+a((3ia'e/ Nn 8r'h+a((3ia'eMi:e/+8r'h+a((3ia'e/


43/46

Ba'3h Gr'h ine'i3(/tationary phase

Cell ly(i( &ay 33$r an/ )ia6le 3ell &a(( &ay /r%4

A (e3n/ 8r'h %ha(e &ay 33$r an/ 3ell( &ay8r n ly(i( %r/$3'( 7 ly(e/ 3ell( *3ry%'i38r'h-

En/8en$( &e'a6li(& 33$r( 6y 3a'a6li=in83ell$lar re(er)e( 7r ne 6$il/in8 6l39( an/ener8y+%r/$3in8 &n&er *&ain'enan3e ener8y-4

The ra'e /e(3ri6in8 'he 3n)er(in 7 3ell &a(( in'&ain'enan3e ener8y r 'he l(( 7 3ell &a(( /$e '3ell ly(i(

.metabolismendogenousforconstantratetheisd

d

k

X k dt

dX −=


44/46

Ba'3h Gr'h ine'i3(

,eath Phase0 The li)in8 r8ani(& %%$la'in /e3rea(e( i'h

'i&e. /$e ' a la39 7 n$'rien'( an/ ':i3&e'a6li3 6y+%r/$3'(4

The ra'e 7 /ea'h $($ally 7ll(

constant.ratedeathorder*first theis'

'

d

d

k

N k dt

dN −=


45/46

Ba'3h Gr'h ine'i3(

"iel/ 3eV3ien'( /e>ne/ 6a(e/ n 'hea&$n' 7 3n($&%'in 7 an'her&a'erial4

)

:o%ygenof nconsumptioon basedyield+ro$th

:yieldroduct

:yield+ro$th

)"

"

"

O

X

Y

S P Y

S

X Y

O X

S P

S X

∆

∆−≡

∆

∆−≡

∆

∆−≡

energy emaintenancenergygro$th

product

lare%tracelluaninto

onassimilati biomassinto

onassimilati S S S S S ∆+∆+∆+∆=∆


46/46

Ba'3h Gr'h ine'i3(

"iel/ 3eV3ien'( /e>ne/ 6a(e/ n 'hea&$n' 7 3n($&%'in 7 an'her &a'erial4

Fr &(' 6a3'eria an/ yea('

":( 04+04 88 8l$3(e

":O204J 14 88 O2

A' 'he en/ 7 'he 6a'3h 8r'h %eri/. 'he &ea($re/ yiel/( area%%aren' a( 7 en/8en$( &e'a6li(& 33$rrin8. / 0.hi3h 3han8e( 'he &e'a6li3 %a'hay( 7 'he ($6('ra'e4e484

App M

Y Y >

HOW CELL GROW&Batch Growth Kinetics

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